Active agent transport systems

ABSTRACT

Methods for transporting a biologically active agent across a cellular membrane or a lipid bilayer. A first method includes the steps of:
         (a) providing a biologically active agent which can exist in a native conformational state, a denatured conformational state, and an intermediate conformational state which is reversible to the native state and which is conformationally between the native and denatured states;   (b) exposing the biologically active agent to a complexing perturbant to reversibly transform the biologically active agent to the intermediate state and to form a transportable supramolecular complex; and   (c) exposing the membrane or bilayer to the supramolecular complex, to transport the biologically active agent across the membrane or bilayer. The perturbant has a molecular weight between about 150 and about 600 daltons, and contains at least one hydrophilic moiety and at least one hydrophobic moiety. The supramolecular complex comprises the perturbant non-covalently bound or complexed with the biologically active agent. In the present invention, the biologically active agent does not form a microsphere after interacting with the perturbant. A method for preparing an orally administrable biologically active agent comprising steps (a) and (b) above is also provided as are oral delivery compositions.       

     Additionally, mimetics and methods for preparing mimetics are contemplated.

This is a continuation of U.S. application Ser. No. 09/420,200, filedOct. 18, 1999; which is a continuation of U.S. application Ser. No.08/763,183, filed Dec. 10, 1996, now U.S. Pat. No. 6,099,856; which is acontinuation-in-part of Ser. No. (a) U.S. Ser. No. 08/328,932, filedOct. 25, 1994, now U.S. Pat. No. 5,714,167; (b) U.S. Ser. No.08/051,019, filed Apr. 22, 1993, now U.S. Pat. No. 5,451,410; (c) U.S.Ser. No. 08/168,776, filed Dec. 16, 1993, now U.S. Pat. No. 5,447,728,which is a continuation-in-part of U.S. Ser. No. 08/051,019, filed Apr.22 1993, now U.S. Pat. No. 5,451,410, and of U.S. Ser. No. 08/143,571,filed Oct. 26, 1993, which is a continuation-in-part of U.S. Ser. No.08/076,803, filed Jun. 14, 1993, now U.S. Pat. No. 5,578,323, which is acontinuation-in-part of U.S. Ser. No 07/920,346, filed Jul. 27, 1992,now U.S. Pat. No. 5,443,841, which is a continuation-in-part of U.S.Ser. No. 07/898,909, filed Jun. 15, 1992; (d) PCT Serial No.PCT/US94/04560, filed Apr. 22, 1994, which is a continuation-in-part ofU.S. Ser. No. 08/051,019, filed Apr. 22, 1993, now U.S. Pat. No.5,451,410, and of U.S. Ser. No. 08/205,511, filed on Mar. 2, 1994, nowU.S. Pat. No. 5,792,451; (e) U.S. Ser. No. 08/231,622, filed Apr. 22,1994, now U.S. Pat. No 5,629,020; (f) U.S. Ser. No. 08/205,511, filedMar. 2, 1994, now U.S. Pat. No. 5,792,451; (g) U.S. Ser. No. 08/231,623,filed Apr. 22, 1994, now U.S. Pat. No. 5,541,155; (h) U.S. Ser. No.08/315,200, filed Sep. 29, 1994, now U.S. Pat. No. 5,693,338; and (i)U.S. Ser. No. 08/316,404, filed Sep. 30, 1994, now U.S. Pat. No.6,331,318. These prior applications are hereby incorporated herein byreference, in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions fortransporting active agents, and particularly biologically active agents,across cell membranes or or lipid bilayers. These methods andcompositions facilitate the delivery of an active agent to a target,such as the delivery of a pharmaceutical agent through an adverseenvironment to a particular location of the body.

BACKGROUND OF THE INVENTION

Conventional means for delivering active agents to their intendedtargets, e.g. human organs, tumor cites, etc., are often severelylimited by the presence of biological, chemical, and physical barriers.Typically, these barriers are imposed by the environment through whichdelivery must take place, the environment of the target for delivery, orthe target itself.

Biologically active agents are particularly vulnerable to such barriers.Oral delivery to the circulatory system for many biologically activeagents would be the route of choice for administration to animals if notfor physical barriers such as the skin, lipid bi-layers, and variousorgan membranes that are relatively impermeable to certain biologicallyactive agents, but which must be traversed before an agent delivered viathe oral route can reach the circulatory system. Additionally, oraldelivery is impeded by chemical barriers such as the varying pH in thegastrointestinal (GI) tract and the presence in the oral cavity and theGI tract of powerful digestive enzymes.

Calcitonin and insulin exemplify the problems confronted in the art indesigning an effective oral drug delivery system. The medicinalproperties of calcitonin and insulin can be readily altered using anynumber of techniques, but their physicochemical properties andsusceptibility to enzymatic digestion have precluded the design of acommercially viable delivery system. Others among the numerous agentswhich are not typically amenable to oral administration are biologicallyactive proteins such as the cytokines (e.g. interferons, IL-2, etc);erythropoietin; polysaccharides, and in particular mucopolysaccharidesincluding, but not limited to, heparin; heparinoids; antibiotics; andother organic substances. These agents are also rapidly renderedineffective or are destroyed in the GI tract by acid hydrolysis,enzymes, or the like.

Biotechnology has allowed the creation of numerous other compounds, ofwhich many are in clinical use around the world. Yet, the current modeof administration of these compounds remains almost exclusively viainjection. While in many cases oral administration of these compoundswould be preferable, these agents are labile to various enzymes andvariations in pH in the GI tract and are generally unable to penetrateadequately the lipid bilayers of which cell membranes are typicallycomposed. Consequently, the active agent cannot be delivered orally tothe target at which the active agent renders its intended biologicaleffect.

Typically, the initial focus of drug design is on the physiochemicalproperties of pharmaceutical compounds and particularly theirtherapeutic function. The secondary design focus is on the need todeliver the active agent to its biological target(s). This isparticularly true for drugs and other biologically active agents thatare designed for oral administration to humans and other animals.However, thousands of therapeutic compounds are discarded because nodelivery systems are available to ensure that therapeutic titers of thecompounds will reach the appropriate anatomical location orcompartment(s) after administration and particularly oraladministration. Furthermore, many existing therapeutic agents areunderutilized for their approved indications because of constraints ontheir mode(s) of administration. Additionally, many therapeutic agentscould be effective for additional clinical indications beyond those forwhich they are already employed if there existed a practical methodologyto deliver them in appropriate quantities to the appropriate biologicaltargets.

Although nature has achieved successful inter- and intra-cellulartransport of active agents such as proteins, this success has not beentranslated to drug design. In nature, the transportable conformation ofan active agent such as a protein is different than the conformation ofthe protein in its native state. In addition, natural transport systemsoften effect a return to the native state of the protein subsequent totransport. When proteins are synthesized by ribosomes, they are shuttledto the appropriate cellular organelle by a variety of mechanisms e.g.signal peptides and/or chaperoning. Gething, M-J., Sambrook, J., Nature,355, 1992, 33-45. One of the many functions of either the signalpeptides or the chaperonins is to prevent premature folding of theprotein into the native state. The native state is usually described asthe 3-dimensional state with the lowest free energy. By maintaining theprotein in a partially unfolded state, the signal peptides or thechaperonins facilitate the protein's ability to cross various cellularmembranes until the protein reaches the appropriate organelle. Thechaperonin then separates from the protein or the signal peptide iscleaved from the protein, allowing the protein to fold to the nativestate. It is well known that the ability of the protein to transitcellular membranes is at least partly a consequence of being in apartially unfolded state.

Current concepts of protein folding suggest that there are a number ofdiscrete conformations in the transition from the native state to thefully denatured state. Baker, D., Agard, D. A., Biochemistry, 33, 1994,7505-7509. The framework model of protein folding suggests that in theinitial early stages of folding the domains of the protein that are thesecondary structure units will form followed by the final folding intothe native state. Kim, P. S., Baldwin, R. L., Annu. Rev. Biochem., 59,1990, 631-660. In addition to these kinetic intermediates, equilibriumintermediates appear to be significant for a number of cellularfunctions. Bychkova, V. E., Berni, R., et al, Biochemistry, 31, 1992,7566-7571, and Sinev, M. A., Razgulyaev, O. I., et al, Eur. J. Biochem.,1989, 180, 61-66. Available data on chaperonins indicate that theyfunction, in part, by keeping proteins in a conformation that is not thenative state. In addition, it has been demonstrated that proteins inpartially unfolded states are able to pass through membranes, whereasthe native state, especially of large globular proteins, penetratesmembranes poorly, if at all. Haynie, D. T., Freire, E.,Proteins:Structure, Function and Genetics, 16, 1993, 115-140.

Similarly, some ligands such as insulin which are unable to undergoconformational changes associated with the equilibrium intermediatesdescribed above, lose their functionality. Hua, Q. X., Ladbury, J. E.,Weiss, M. A., Biochemistry, 1993, 32, 1433-1442; Remington, S., Wiegand,G., Huber, R., 1982, 158, 111-152; Hua, Q. X., Shoelson, S. E.,Kochoyan, M. Weiss, M. A., Nature, 1991, 354, 238-241.

Studies with diphtheria toxin and cholera toxin indicate that afterdiphtheria toxin binds to its cellular receptor, it is endocytosed, andwhile in this endocytic vesicle, it is exposed to an acidic pHenvironment. The acidic pH induces a structural change in the toxinmolecule which provides the driving force for membrane insertion andtranslocation to the cytosol. See, Ramsay, G., Freire, E. Biochemistry,1990, 29, 8677-8683 and Schon, A., Freire, E., Biochemistry, 1989, 28,5019-5024. Similarly, cholera toxin undergoes a conformational changesubsequent to endocytosis which allows the molecule to penetrate thenuclear membrane. See also, Morin, P. E., Diggs, D., Freire, E.,Biochemistry, 1990, 29, 781-788.

Earlier designed delivery systems have used either an indirect or adirect approach to delivery. The indirect approach seeks to protect thedrug from a hostile environment. Examples are enteric coatings,liposomes, microspheres, microcapsules. See, colloidal drug deliverysystems, 1994, ed. Jorg Freuter, Marcel Dekker, Inc.; U.S. Pat. No.4,239,754; Patel et al. (1976), FEBS Letters, Vol. 62, pg. 60; andHashimoto et al. (1979), Endocrinology Japan, Vol. 26, pg. 337. All ofthese approaches are indirect in that their design rationale is notdirected to the drug, but rather is directed to protecting against theenvironment through which the drug must pass enroute to the target atwhich it will exert its biological activity, i.e. to prevent the hostileenvironment from contacting and destroying the drug.

The direct approach is based upon forming covalent linkages with thedrug and a modifier, such as the creation of a prodrug. Balant, L. P.,Doelker, E., Buri, P., Eur. J. Drug Metab. And Pharmacokinetics, 1990,15(2), 143-153. The linkage is usually designed to be broken underdefined circumstances, e.g. pH changes or exposure to specific enzymes.The covalent linkage of the drug to a modifier essentially creates a newmolecule with new properties such as an altered log P value and/or aswell as a new spatial configuration. The new molecule has differentsolubility properties and is less susceptible to enzymatic digestion. Anexample of this type of method is the covalent linkage of polyethyleneglycol to proteins. Abuchowski, A., Van Es, T., Palczuk, N. C., Davis,F. F., J. Biol. Chem. 1977, 252, 3578.

Broad spectrum use of prior delivery systems has been precluded,however, because: (1) the systems require toxic amounts of adjuvants orinhibitors; (2) suitable low molecular weight cargos, i.e. activeagents, are not available; (3) the systems exhibit poor stability andinadequate shelf life; (4) the systems are difficult to manufacture; (5)the systems fail to protect the active agent (cargo); (6) the systemsadversely alter the active agent; or (7) the systems fail to allow orpromote absorption of the active agent.

There is still a need in the art for simple, inexpensive deliverysystems which are easily prepared and which can deliver a broad range ofactive agents to their intended targets, expecially in the case ofpharmaceutical agents that are to be administered via the oral route.

SUMMARY OF THE INVENTION

The present invention discloses methods for transporting a biologicallyactive agent across a cellular membrane or a lipid bilayer. A firstmethod includes the steps of:

-   -   (a) providing a biologically active agent which can exist in a        native conformational state, a denatured conformational state,        and an intermediate conformational state which is reversible to        the native state and which is conformationally between the        native and denatured states;    -   (b) exposing the biologically active agent to a complexing        perturbant to reversibly transform the biologically active agent        to the intermediate state and to form a transportable        supramolecular complex; and    -   (c) exposing the membrane or bilayer to the supramolecular        complex, to transport the biologically active agent across the        membrane or bilayer.

The perturbant has a molecular weight between about 150 and about 600daltons, and contains at least one hydrophilic moiety and at least onehydrophobic moiety. The supramolecular complex comprises the perturbantnon-covalently bound or complexed with the biologically active agent. Inthe present invention, the biologically active agent does not form amicrosphere after interacting with the perturbant.

Also contemplated is a method for preparing an orally administrablebiologically active agent comprising steps (a) and (b) above.

In an alternate embodiment, an oral delivery composition is provided.The composition comprises a supramolecular complex including:

-   -   (a) a biologically active agent in an intermediate        conformational state which is reversible to the native state,        non-covalently complexed with    -   (b) a complexing perturbant having a molecular weight ranging        from about 150 to about 600 and having at least one hydrophilic        moiety and at least one hydrophobic moiety;        -   wherein the intermediate state is conformationally between            the native conformation state and denatured conformation            state of the biologically active agent and the composition            is not a microsphere.

Further contemplated is a method for preparing a mimetic which istransportable across cellular membrane(s) or lipid-bilayer(s) and whichis bioavailable to the host after crossing the membrane(s) orbilayer(s). A biologically active agent which can exist in a nativeconformational state, a denatured conformational state, and anintermediate conformational state which is reversible to the nativestate and which is conformationally between the native state and thedenatured state, is exposed to a complexing perturbant to reversiblytransform the biologically active agent to the intermediateconformational state and to form a transportable supramolecular complex.The perturbant has a molecular weight between about 150 and about 600daltons and at least one hydrophilic moiety and one hydrophilic moiety.The supramolecular complex comprises the perturbant non-covalentlycomplexed with the biologically active agent, and the biologicallyactive agent does not form a microsphere with the perturbant. A mimeticof the supramolecular complex is prepared.

Alternatively, a method for preparing an agent which is transportableacross a cellular membrane or a lipid-bilayer, and which is bioavailableafter crossing the membrane or bilayer, is provided. A biologicallyactive agent which can exist in a native conformational state, adenatured conformational state, and an intermediate conformational statewhich is reversible to the native state and which is conformationallybetween the native and denatured states, is exposed to a perturbant toreversibly transform the biologically active agent to the intermediatestate. The agent, a mimetic of the intermediate state, is prepared.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is illustration of a native gradient gel of α-interferon (IFN)and a modified amino acid complexing perturbant.

FIG. 2 is an illustration of a native gradient gel of α-interferon and athermal condensate complexing perturbant.

FIG. 3 is a graphic illustration of serum levels of α-interferon afteroral administration of α-interferon with and without complexingperturbants.

FIG. 4 is a graphic illustration of changes in serum calcium in ratsorally administered salmon calcitonin with and without complexingperturbants.

FIG. 5 is a graphic illustration of guanidine hydrochloride (GuHCl)induced denaturation of α-interferon.

FIG. 6 is a graphic illustration of the concentration effect of GuHCl onα-interferon conformation.

FIG. 7 is a graphic illustration of the pH denaturation of α-interferon.

FIG. 8 is a graphic illustration of the pH denaturation of insulin.

FIGS. 9A and 9B are graphic illustrations of the reversibility of thecircular dichroism spectrum of α-interferon.

FIG. 10 is a graphic illustration of the circular dichroism spectrum ofα-interferon.

FIG. 11 is a graphic illustration of intrinsic tryptophan fluorescenceof α-interferon and a complexing perturbant.

FIG. 12 is a graphic illustration of serum levels of α-interferon afteroral administration of α-interferon with and without complexingperturbant.

FIG. 13 is a graphic illustration of the differential scanningcalorimetry of α-interferon and complexing perturbant.

FIGS. 14A and 14B are graphic illustrations of the reversibility of thetransformation due to complexing perturbants.

FIG. 15 is a graphic illustration of the effect of complexing perturbanton α-interferon.

FIG. 16 is a graphic illustration of serum levels of α-interferon afteroral administration of α-interferon with and without complexingperturbant.

FIG. 17 is a graphic illustration of the concentration effect ofcomplexing perturbant on α-interferon conformation.

FIG. 18 is a graphic illustration of serum levels of α-interferon afteroral administration with and without complexing perturbant.

FIG. 19 is a graphic illustration of the effect of complexing perturbanton α-interferon.

FIG. 20 is a graphic illustration of the Isothermal TitrationCalorimetry of α-interferon and complexing perturbant.

FIG. 21 is a graphic illustration of the Isothermal TitrationCalorimetry of α-interferon and complexing perturbant.

FIG. 22 is a graphic illustration of the effects of complexingperturbants on α-interferon.

FIG. 23 is a graphic illustration of the effect of the concentration ofcomplexing perturbants on α-interferon.

FIG. 24 is a graphic illustration of the Isothermal TitrationCalorimetry of α-interferon and complexing perturbant.

FIG. 25 is a graphic illustration of serum levels of α-interferon afteroral administration with complexing perturbants.

FIG. 26 is a graphic illustration of the in vivo pharmacokinetics ofrecombinant human growth hormone mixed with complexing perturbants.

FIG. 27 is a graphic illustration of pancreative inhibition assay withα-interferon and complexing perturbants.

FIG. 28 is a graphic illustration of the effect of DSC of heparin at pH5.0.

FIG. 29 is a graphic illustration of the degree of retardation vs. peakAPTT values from in vivo dosing experiments with heparin.

FIG. 30 is a graphic illustration of clotting time in rats after oraladministration of heparin with and without complexing perturbants.

FIG. 31 is a graphic illustration of the DSC of DPPC with perturbantcompounds at several concentrations (units T_(M), ° C. vs.concentration).

FIG. 32 is a graphic illustration of the concentration effect ofcomplexing perturbant compound L on the DPPC conformation.

FIG. 33 is a graphic illustration of the concentration effect ofcomplexing perturbant compound L on rhGH conformation.

FIG. 34 is a graphic illustration of the concentration effect ofcomplexing perturbant compound LI on rhGH conformation.

FIG. 35 is a graphic illustration of the concentration effect ofcomplexing perturbant compound XI on rhGH conformation.

FIG. 36 is a graphic illustration of the differential light scatteringof perturbant compound L in a 10 mM phosphate buffer at pH 7.0.

DETAILED DESCRIPTION OF THE INVENTION

All biological organisms can be described as being comprised of aqueouscompartments separated from one another by cell membranes or lipidbilayers. Active agents, and particularly pharmacologic or therapeuticactive agents, have one solubility value in an aqueous environment andan entirely different solubility value in a hydrophobic environment.Typically, delivery of an active agent from the site of administrationto the target site, such as a site of pathology, requires passing theactive agent through cell membranes or lipid bilayers in which thesolubility of the active agent will vary. Additionally, oral delivery ofactive agent requires the ability to resist enzymatic degradation, pHdifferentials, and the like. These barriers result in significantirreversible partial, or in some instances total, loss of the activeagent or its biological activity between the site of administration andthe target. Consequently, the quantity of active agent that is requiredto elicit a proper response, such as a therapeutic response, may notreach the target. Therefore, active agents require some assistance inreaching and then in crossing these membranes or lipid bilayers.

The present invention effects active agent delivery by creating areversibly non-covalently complexed supramolecule from the active agentand complexing perturbant. As a result, the three-dimensional structureor conformation of the active agent is changed, but the chemicalcomposition of the active agent molecule is not altered. This alterationin structure (but not composition) provides the active agent with theappropriate solubility (log P) to cross or penetrate the membrane orlipid bilayer and to resist enzymatic degradation and the like. Crossingrefers to transport from one side of the cell membrane or lipid bilayerto the opposite side (i.e. from the outside or exterior to the inside orinterior of a cell and/or visa versa), whether the cell membrane orlipid bilayer is actually penetrated or not. Additionally, the perturbedintermediate state of the active agent or the supramolecular complexitself can be used as a template for the preparation of mimetics whichwould, accordingly, be transportable across a cell membrane or a lipidbilayer. After crossing the cell membrane or lipid bilayer, an activeagent has biological activity and bioavailability, either by restorationto the native state or by retaining biological activity orbioavailability acquired in the intermediate state. The mimetic actssimilarly after crossing the cell membrane or lipid bilayer.

Active Agents

The native conformational state of an active agent is typicallydescribed as the three dimensional state with the lowest free energy(ΔG). It is the state in which the active agent typically possesses thefull complement of activity ascribed to the agent, such as the fullcomplement of biological activity ascribed to a biologically activeagent.

The denatured conformational state is the state in which the activeagent has no secondary or tertiary structure.

Intermediate conformational states exist between the native anddenatured states. A particular active agent may have one or moreintermediate states. The intermediate state achieved by the presentinvention is structurally and energetically distinct from both thenative and denatured states. Active agents useful in the presentinvention must be transformable from their native conformational stateto a transportable intermediate conformational state and back to theirnative state, i.e. reversibly transformable, so that when the activeagent reaches its target, such as when an orally delivered drug reachesthe circulatory system, the active agent retains, regains, or acquires abiologically, pharmacologically, or therapeutically significantcomplement of its desired biological activity. Preferably the ΔG of theintermediate state ranges from about −20 Kcal/mole to about 20Kcal/mole, and most preferably, it ranges from about −10 Kcal/mole toabout 10 Kcal/mole.

For example in the case of a protein, the intermediate state hassignificant secondary structure, significant compactness due to thepresence of a sizable hydrophobic core, and a tertiary structurereminiscent of the native fold but without necessarily exhibiting thepacking of the native state. The difference in free energy (ΔG) betweenthe intermediate state and the native state is relatively small. Hence,the equilibrium constant between the native and the transportable,reversible intermediate state(s) is close to unity (depending uponexperimental conditions). Intermediate states can be confirmed by, forexample, differential scanning calorimetry (DSC), isothermal titrationcalorimetry (ITC), native gradient gels, NMR, fluorescence, and thelike.

Without being bound by any theory, applicants believe that the physicalchemistry of the intermediate state can be understood by the followingexplanation relating to proteinaceous active agents. Proteins can existin stable intermediate conformations that are structurally andenergetically distinct from either the native state or the denaturedstate. The inherent stability of any conformation(s) of any protein isreflected in the Gibbs free energy of the conformation(s). The Gibbsfree energy for any state of a monomeric protein is describedthermodynamically by the following relationship:ΔG°=ΔH°(T _(R))−TΔS°(T _(R))+ΔCp°((T−T _(R))−T In(T/T _(R)))  (1)where T is the temperature, T_(R) is a reference temperature, ΔH°(T_(R))and TΔS°(T_(R)) are the relative enthalpy and entropy of this state atthe reference temperature, and ΔCp° is the relative heat capacity ofthis state. It is convenient to chose the native state as the referencestate to express all relative thermodynamic parameters.

The sum of the statistical weights of all states accessible to theprotein is defined as the partition function Q: $\begin{matrix}{Q = {\sum\limits_{l = 0}^{n}{\mathbb{e}}^{{- \Delta}\quad{{Gi}/{RT}}}}} & (2)\end{matrix}$Equation 2 can also be written as: $\begin{matrix}{Q = {1 + {\sum\limits^{n - 1}{\mathbb{e}}^{{- {DGi}}/{RT}}} + {\mathbb{e}}^{{- {DGn}}/{RT}}}} & (3)\end{matrix}$where the second term includes all the intermediates that becomepopulated during the transition. The first and last terms of equation(3) are the statistical weights of the native and denatured states,respectively. Under most conditions, protein structure could beapproximated by a two-state transition function:Q≈1+e ^(−ΔGn/RT)  (4)

See, Tanford, C., Advances in Protein Chemistry, 1968, 23, 2-95.Conformations of proteins that are intermediate between the native stateand the denatured state can be detected by, for example, NMR,calorimetry, and fluorescence. Dill, K. A., Shortle, D., Annu. Rev.Biochem. 60, 1991, 795-825.

All thermodynamic parameters can be expressed in terms of the partitionfunction. Specifically the population of molecules in state i is givenin equation (5): $\begin{matrix}{{Pi} = \frac{{\mathbb{e}}^{{- \Delta}\quad{{Gi}/{RT}}}}{Q}} & (5)\end{matrix}$Therefore, measurement of the appropriate terms in equation (1) thatwould allow for the calculation of the Gibbs free energy would determinethe extent to which any intermediate state(s) is populated to anysignificant degree under defined experimental conditions. This in turnindicates the role that these intermediate state(s) play in drugdelivery. The more populated the intermediate state, the more efficientthe delivery.

Active agents suitable for use in the present invention includebiologically active agents and chemically active agents, including, butnot limited to, fragrances, as well as other active agents such as, forexample, cosmetics.

Biologically active agents include, but are not limited to, pesticides,pharmacological agents, and therapeutic agents. For example,biologically active agents suitable for use in the present inventioninclude, but are not limited to, peptides, and particularly smallpeptides; hormones, and particularly hormones which by themselves do notor only pass slowly through the gastro-intestinal mucosa and/or aresusceptible to chemical cleavage by acids and enzymes in thegastro-intestinal tract; polysaccharides, and particularly mixtures ofmuco-polysaccharides; carbohydrates; lipids; or any combination thereof.Further examples include, but are not limited to, human growth hormones;bovine growth hormones; growth releasing hormones; interferons;interleukin-1; insulin; heparin, and particularly low molecular weightheparin; calcitonin; erythropoletin; atrial naturetic factor; antigens;monoclonal antibodies; somatostatin; adrenocorticotropin, gonadotropinreleasing hormone; oxytocin; vasopressin; cromolyn sodium (sodium ordisodium chromoglycate); vancomycin; desferrioxamine (DFO);anti-microbials, including, but not limited to anti-fungal agents; orany combination thereof.

The methods and compositions of the present invention may combine one ormore active agents.

Perturbants

Perturbants serve two purposes in the present invention. In a firstembodiment, the active agent is contacted with a perturbant whichreversibly transforms the active agent from the native state to theintermediate transportable state. The perturbant non-covalentlycomplexes with the active agent to form a supramolecular complex whichcan permeate or cross cell membranes and lipid bilayers. Thissupramolecular complex can be used as a template for the design of amimetic or can be used as a delivery composition itself. The perturbant,in effect, fixes the active agent in the transportable intermediatestate. The perturbant can be released from the supramolecular complex,such as by dilution in the circulatory system, so that the active agentcan return to the native state. Preferably, these perturbants have atleast one hydrophilic (i.e. readily soluble in water, such as forexample, a caroxylate group) and at least one hydrophobic moiety (i.e.readily soluble in an orginac solvent such as, for example, a benzenegroup), and have a molecular weight ranging from about 150 to about 600daltons and most preferably from about 200 to about 500 daltons.

Complexing perturbant compounds include, but are not limited toproteinoids including linear, non-linear, and cyclic proteinoids;modified (acylated or sulfonated) amino acids, poly amino acids, andpeptides; modified amino acid, poly amino acid, or peptide derivatives(ketones or aldehydes); diketopiperazine/amino acid constructs;carboxylic acids; and various other perturbants discussed below.

Again without being bound by any theory, applicant believes that thenon-covalent complexing may be effected by intermolecular forcesincluding but not limited to, hydrogen bonding, hydrophilicinteractions, electrostatic interactions, and Van der Waalsinteractions. For any given active agent/perturbant supramolecularcomplex, there will exist some combination of the aforementioned forcesthat maintain the association.

The association constant K_(a) between the perturbant and the activeagent can be defined according to equation (6)Ka=e ^(−ΔG/RT)  (6)

The dissociation constant K_(d) is the reciprocal of K_(a). Thusmeasurement of the association constants between perturbant and activeagent at a defined temperature will yield data on the molar Gibbs freeenergy which allows for the determination of the associated enthalpicand entropic effects.

Experimentally these measurements can be made, for example, using NMR,fluorescence or calorimetry.

This hypothesis can be illustrated with proteins in the followingmanner:Protein unfolding can be described according to the equilibrium thatexists between its various conformational states, e.g.

where N is the native state, I is the intermediate state(s), D is thedenatured state, and k₁ and k₂ are the respective rate constants. K₁ andK₂ are the respective equilibrium constants. Accordingly,$\quad\begin{matrix}\begin{matrix}{Q = {\sum\limits_{i = 0}^{n}{\mathbb{e}}^{{- \Delta}\quad{G/{RT}}}}} \\{= {1 + {\mathbb{e}}^{{- \Delta}\quad{G_{1}/{RT}}} + {\mathbb{e}}^{{- \Delta}\quad{G_{2}/{RT}}}}} \\{= {1 + K_{1} + {K_{2}(8)}}} \\{= {1 + k_{1} + {k_{1}{k_{2}(9)}}}}\end{matrix} & (2)\end{matrix}$

This suggests that increasing the partition function of the intermediatestate(s) should have a positive impact on the ability to deliver theactive agent, i.e. $\begin{matrix}{P_{I} = \frac{K_{1}}{\left( {1 + K_{1} + K_{2}} \right)}} & (10)\end{matrix}$

Because complexing must be reversible, the complexing of the perturbantwith the active agent, as measured by the K_(a), must be strong enoughto insure delivery of the drug either to the systemic circulation and/orto the target(s), but not so strong so that disengagement of theperturbant will not occur in a timely manner to allow the active agentto renature if necessary to produce the desired effect(s).

In a second embodiment, perturbants reversibly transform the activeagent to the intermediate state so that the conformation of that statecan be used as a template for the preparation of mimetics. Perturbantsfor this purpose need not, but may, complex with the active agent.Therefore, in addition to the complexing perturbants discussed above,perturbants that change the pH of the active agent or its environment,such as for example, strong acids or strong bases; detergents;perturbants that change the ionic strength of the active agent or itsenvironment; other agents such as for example, guanidine hydrochloride;and temperature can be used to transform the active agent. Either thesupramolecular complex or the reversible intermediate state can be usedas a template for mimetic design.

Complexing Perturbants

Amino acids are the basic materials used to prepare many of thecomplexing perturbants useful in the present invention. An amino acid isany carboxylic acid having at least one free amine group and includesnaturally occurring and synthetic amino acids. The preferred amino acidsfor use in the present invention are ∝-amino acids, and most preferablyare naturally occurring ∝-amino acids. Many amino acids and amino acidesters are readily available from a number of commercial sources such asAldrich Chemical Co. (Milwaukee, Wis., USA); Sigma Chemical Co. (St.Louis, Mo., USA); and Fluka Chemical Corp. (Ronkonkoma, N.Y., USA).

Representative, but not limiting, amino acids suitable for use in thepresent invention are generally of the formula:

wherein:

-   -   R¹ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;    -   R² is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀        alkenyl) phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl)        naphthyl, phenyl (C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl),        naphthyl (C₁-C₁₀ alkyl), or naphthyl (C₂-C₁₀ alkenyl);    -   R² being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl,        C₁-C₄ alkoxy, —OH, —SH, —CO₂R³, C₃-C₁₀ cycloalkyl, C₃-C₁₀        cycloalkenyl, heterocycle having 3-10 ring atoms wherein the        hetero atom is one or more of N, O, S, or any combination        thereof, aryl, (C₁-C₁₀ alk)aryl, ar(C₁-C₁₀ alkyl) or any        combination thereof;    -   R² being optionally interrupted by oxygen, nitrogen, sulfur, or        any combination thereof; and    -   R³ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

The preferred naturally occurring amino acids for use in the presentinvention as amino acids or components of a peptide are alanine,arginine, asparagine, aspartic acid, citrulline, cysteine, cystine,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,ornithine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, hydroxy proline, γ-carboxyglutamate, phenylglycine, orO-phosphoserine. The preferred amino acids are arginine, leucine,lysine, phenylalanine, tyrosine, tryptophan, valine, and phenylglycine.

The preferred non-naturally occurring amino acids for use in the presentinvention are β-alanine, α-amino butyric acid, γ-amino butyric acid,γ-(aminophenyl) butyric acid, α-amino isobutyric acid, citrulline,ε-amino caproic acid, 7-amino heptanoic acid, β-aspartic acid,aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid,γ-glutamic acid, cysteine (ACM), ε-lysine, ε-lysine (A-Fmoc), methioninesulfone, norleucine, norvaline, ornithine, d-ornithine,p-nitro-phenylalanine, hydroxy proline,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, and thioproline.

Poly amino acids are either peptides or two or more amino acids linkedby a bond formed by other groups which can be linked, e.g., an ester,anhydride or an anhydride linkage. Special mention is made ofnon-naturally occurring poly amino acids and particularly non-naturallyoccurring hetero-poly amino acids, i.e. of mixed amino acids.

Peptides are two or more amino acids joined by a peptide bond. Peptidescan vary in length from di-peptides with two amino acids to polypeptideswith several hundred amino acids. See, Walker, Chambers BiologicalDictionary, Cambridge, England: Chambers Cambridge, 1989, page 215.Special mention is made of non-naturally occurring peptides andparticularly non-naturally occurring peptides of mixed amino acids.Special mention is also made of di-peptides tri-peptides,tetra-peptides, and penta-peptides, and particularly, the preferredpeptides are di-peptides and tri-peptides. Peptides can be homo- orhetero- peptides and can include natural amino acids, synthetic aminoacids, or any combination thereof.

Proteinoid Complexing Perturbants

Proteinoids are artificial polymers of amino acids. The proteinoidspreferably are prepared from mixtures of amino acids. Preferredproteinoids are condensation polymers, and most preferably, are thermalcondensation polymers. These polymers may be directed or randompolymers. Proteinoids can be linear, branched, or cyclical, and certainproteinoids can be units of other linear, branched, or cyclicalproteinoids.

Special mention is made of diketopiperazines. Diketopiperizines are sixmember ring compounds. The ring includes two nitrogen atoms and issubstituted at two carbons with two oxygen atoms. Preferably, thecarbonyl groups are at the 1 and 4 ring positions. These rings can beoptionally, and most often are, further substituted.

Diketopiperazine ring systems may be generated during thermalpolymerization or condensation of amino acids or amino acid derivatives.(Gyore, J; Ecet M. Proceedings Fourth ICTA (Thermal Analysis), 1974, 2,387-394 (1974)). These six membered ring systems were presumablygenerated by intra-molecular cyclization of the dimer prior to furtherchain growth or directly from a linear peptide (Reddy, A. V., Int. J.Peptide Protein Res., 40, 472-476 (1992); Mazurov, A. A. et al., Int. J.Peptide Protein Res., 42, 14-19 (1993)).

Diketopiperazines can also be formed by cyclodimerization of amino acidester derivatives as described by Katchalski et al., J. Amer. Chem.Soc., 68, 879-880 (1946), by cyclization of dipeptide ester derivatives,or by thermal dehydration of amino acid derivatives and high boilingsolvents as described by Kopple et al., J. Org. Chem., 33 (2), 862-864(1968).

In a typical synthesis of a diketopiperazine, the COOH group(s) of anamino acid benzyl ester are activated in a first step to yield aprotected ester. The amine is deprotected and cyclized via dimerizationin a second step, providing a diketopiperazine di-ester. Finally, theCOOH group(s) are deprotected to provide the diketopiperazine.

Diketopiperazines typically are formed from α-amino acids. Preferably,the α-amino acids of which the diketopiperazines are derived areglutamic acid, aspartic acid, tyrosine, phenylalanine, and opticalisomers of any of the foregoing.

Special mention is made of diketopiperizines of the formula:

wherein R⁴, R⁵, R⁶, and R⁷ independently are hydrogen, C₁-C₂₄ alky,C₁-C₂₄ alkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl)phenyl, (C₁-C₁₀alkenyl)phenyl, (C₁-C₁₀ alkyl)naphthyl, (C₁-C₁₀ alkenyl)naphthyl, phenyl(C₁-C₁₀ alkyl), phenyl(C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), andnaphthyl (C₁-C₁₀ alkenyl); any of R⁴, R⁵, R⁶, and R⁷ independently mayoptionally be substituted with C₁-C₄ alkyl, C₁-C₄ alkenyl, C₁-C₄ alkoxy,—OH, —SH, and —CO₂R⁸ or any combination thereof; R⁸ is hydrogen, C₁-C₄alkyl or C₁-C₄ alkenyl; and any of R⁴, R⁵, R⁶, and R⁷ independently mayoptionally be interrupted by oxygen, nitrogen, sulfur, or anycombination thereof.

The phenyl or naphthyl groups may optionally be substituted. Suitable,but non-limiting, examples of substituents are C₁-C₆ alkyl, C₁-C₆alkenyl, C₁-C₆ alkoxy, —OH, —SH, or CO₂R⁹ wherein R⁹ is hydrogen, C₁-C₆alkyl, or C₁-C₆ alkenyl.

Preferably, R⁶ and R⁷ independently are hydrogen, C₁-C₄ alkyl or C₁-C₄alkenyl. Special mention is made of diketopiperazines which arepreferred complexing perturbants. These diketopiperazines include theunsubstituted diketopiperazine in which R⁴, R⁵, R⁶, and R⁷ are hydrogen,and diketopiperazines which are substituted at one or both of thenitrogen atoms in the ring, i.e. mono or di-N-substituted. Specialmention is made of the N-substituted diketopiperazine wherein one orboth of the nitrogen atoms is substituted with a methyl group.

Special mention is also made of diketopiperizines of the formula

wherein R¹⁰ and R¹¹ independently are hydrogen, C₁-C₂₄ alkyl, C₁-C₂₄alkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl)phenyl, (C₁-C₁₀ alkenyl)phenyl,(C₁-C₁₀ alkyl)naphthyl, (C₁-C₁₀ alkenyl)naphthyl, phenyl (C₁-C₁₀ alkyl),phenyl(C₁-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), and naphthyl (C₁-C₁₀alkenyl); but both R¹⁰ and R¹¹ can not be hydrogen; either or both R¹⁰or R¹¹ independently may optionally be substituted with C₁-C₄ alkyl,C₁-C₄ alkenyl, C₁-C₄ alkoxy, —OH, —SH, and —CO₂R¹² or any combinationthereof; R¹² is hydrogen, C₁-C₄ alkyl or C₁-C₄ alkenyl; and either orboth R¹⁰ and R¹¹ independently may optionally be interrupted by oxygen,nitrogen, sulfur, or any combination thereof.

The phenyl or naphthyl groups may optionally be substituted. Suitable,but non-limiting, examples of substituents are C₁-C₆ alkyl, C₁-C₆,alkenyl, C₁-C₆ alkoxy, —OH, —SH, or CO₂R¹³ wherein R¹³ is hydrogen,C₁-C₆ alkyl, or C₁-C₆ alkenyl. When one of R¹⁰ or R¹¹ is hydrogen, thediketopiperazine is mono-carbon-(C)-substituted. When neither R¹⁰ norR¹¹ is hydrogen, the diketopiperazine is di-carbon-(C)-substituted.

Preferably, R¹⁰, R¹¹, or both R¹⁰ and R¹¹, contain at least onefunctional group, a functional group being a non-hydrocarbon portionresponsible for characteristic reactions of the molecule. Simplefunctional groups are heteroatoms including, but not limited tohalogens, oxygen, sulfur, nitrogen, and the like, attached to, thecarbon of an alkyl group by a single or multiple bond. Other functionalgroups include, but are not limited to, for example, hydroxyl groups,carboxyl groups, amide groups, amine groups, substituted amine groups,and the like.

Preferred diketopiperazines are those which are substituted at one ortwo of the carbons of the ring with a functional group that includes atleast one carboxyl functionality.

Amino Acid(s)/Diketopiperazine Complexing Perturbants

Diketopiperizines may also be polymerized with additional amino acids toform constructs of at least one amino acid or an ester or an amidethereof and at least one diketopiperazine, preferably covalently bondedto one another.

When the diketopiperazine is polymerized with additional amino acids,one or more of the R groups must contain at least one functional group,a functional group being a non-hydrocarbon portion responsible forcharacteristic reactions of the molecule. Simple functional groups areheteroatoms including, but not limited to halogens, oxygen, sulfur,nitrogen, and the like, attached to, the carbon of an alkyl group by asingle or multiple bond. Other functional groups include, but are notlimited to, for example, hydroxyl groups, carboxyl groups, amide groups,amine groups, substituted amine groups, and the like.

Special mention is also made of diketopiperazines which are preferredcomponents of the amino acids/diketopiperazine perturbants of thepresent invention. Such preferred diketopiperazines are those which aresubstituted at one or two of the carbons of the ring and preferably aresubstituted with a functional group that includes at least one carboxylfunctionality.

Most preferably, the diketopiperazines in the aminoacids/diketopiperazine perturbants are prepared from trifunctional aminoacids such as L-glutamic acid and L-aspartic acid which cyclize to formdiketopiperazines.

The diketopiperazines can generate a bis-carboxylic acid platform whichcan be further condensed with other amino acids to form the perturbant.Typically, the diketopiperazine will react and covalently bond with oneor more of the amino acids through the functional group(s) of the Rgroups of the diketopiperazines. These unique systems, because of thecis-geometry imparted by the chiral components of the diketopiperazinering (Lannom, H. K. et al., Int. J. Peptide Protein Res., 28, 67-78(1986)), provide an opportunity to systematically alter the structure ofthe terminal amino acids while holding the orientation between themfixed relative to non-cyclic analogs (Fusaoka et al., Int. J. PeptideProtein Res., 34, 104-110 (1989); Ogura, H. et al., Chem. Pharma. Bull.,23, 2474-2477 (1975). See also, Lee, B. H. et al. J. Org. Chem., 49,2418-2423 (1984); Buyle, R., Helv. Chim. Acta, 49, 1425, 1429 (1966).Other methods of polymerization known to those skilled in the art maylend themselves to amino acid/diketopiperazine polymerization as well.

The amino acids/diketopiperazine perturbants may include one or more ofthe same or different amino acids as well as one or more of the same ordifferent diketopiperazines as described above.

Ester and amide derivatives of these amino acids/diketopiperazineperturbants are also useful in the present invention.

Modified Amino Acid Complexing Perturbants

Modified amino acids, poly amino acids or peptides are either acylatedor sulfonated and include amino acid amides and sulfonamides.

Acylated Amino Acid Complexing Perturbants

Special mention is made of acylated amino acids having the formula:Ar—Y—(R¹⁴)_(n)—OH  IVwherein Ar is a substituted or unsubstituted phenyl or naphthyl;

has the formula

R¹⁵ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl) phenyl, (C₁ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl,(C₁ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ toC₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀alkenyl);

R¹⁵ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁to C₄ alkoxy, —OH, —SH and —CO₂R⁵, cycloalkyl, cycloalkenyl,heterocyclic alkyl, alkaryl, heteroaryl, heteroalkaryl, or anycombination thereof;

R¹⁷ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R¹⁵ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof; and

R¹⁶ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Special mention is also made of those having the formula:

wherein:

R¹⁸ is (i) C₃-C₁₀ cycloalkyl, optionally substituted with C₁-C₇ alkyl,C₂-C₇ alkenyl, C₁-C₇ alkoxy, hydroxy, phenyl, phenoxy or —CO₂R²¹,wherein R¹ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl; or

-   -   (ii) C₁-C₆ alkyl substituted with C₃-C₁₀ cycloalkyl;

R¹⁹ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R²⁰ is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl)phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl(C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl) ornaphthyl (C₂-C₁₀ alkenyl);

R²⁰ being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄alkoxy, —OH, —SH, —CO₂R²², C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,heterocycle having 3-10 ring atoms wherein the hetero atom is one ormore of N, O, S or any combination thereof, aryl, (C₁-C₁₀alk)aryl,ar(C₁-C₁₀ alkyl), or any combination thereof;

R²⁰ being optionally interrupted by oxygen, nitrogen, sulfur, or anycombination thereof; and

R²² is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

Some preferred acylated amino acids include salicyloyl phenylalanine,and the compounds having the formulas:

Special mention is made of compounds having the formula:

wherein A is Try, Leu, Arg, Trp, or Cit; and

optionally wherein if A is Try, Arg, Trp or Cit; A is acylated at 2 ormore functional groups.

Preferred compounds are those wherein A is Try; A is Tyr and is acylatedat 2 functional groups; A is Leu; A is Arg; A is Arg and is acylated at2 functional groups; A is Trp; A is Trp and is acylated at 2 functionalgroups; A is Cit; and A is Cit and is acylated at 2 functional groups.

Special mention is also made of compounds having the formula:

wherein A is Arg or Leu; andwherein if A is Arg, A is optionally acylated at 2 or more functionalgroups;

where A is Leu or phenylglycine;

wherein A is phenylglycine; and

wherein A is phenylglycine.

Acylated amino acids may be prepared by reacting single amino acids,mixtures of two or more amino acids, or amino acid esters with an aminemodifying agent which reacts with free amino moieties present in theamino acids to form amides.

Suitable, but non-limiting, examples of acylating agents useful inpreparing acylated amino acids include acid chloride acylating agentshaving the formula

R²³ an appropriate group for the modified amino acid being prepared,such as, but not limited to, alkyl, alkenyl, cycloalkyl, or aromatic,and particularly methyl, ethyl, cyclohexyl, cyclophenyl, phenyl, orbezyl, and X is a leaving group. Typical leaving groups include, but arenot limited to, halogens such as chlorine, bromine and iodine.

Examples of the acylating agents include, but are not limited to, acylhalides including, but not limited to, acetyl chloride, propyl chloride,cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoylchloride, benzoyl chloride, hippuryl chloride and the like; andanhydrides, such as acetic anhydride, propyl anhydride, cyclohexanoicanhydride, benzoic anhydride, hippuric anhydride and the like. Preferredacylating agents include benzoyl chloride, hippuryl chloride, acetylchloride, cyclohexanoyl chloride, cyclopentanoyl chloride, andcycloheptanoyl chloride.

The amine groups can also be modified by the reaction of a carboxylicacid with coupling agents such as the carbodiimide derivatives of aminoacids, particularly hydrophilic amino acids such as phenylalanine,tryptophan, and tyrosine. Further examples includedicyclohexylcarbodiimide and the like.

If the amino acid is multifunctional, i.e. has more than one —OH, —NH₂or —SH group, then it may optionally be acylated at one or morefunctional groups to form, for example, an ester, amide, or thioesterlinkage.

For example, in the preparation of many acylated amino acids, the aminoacids are dissolved in an aqueous alkaline solution of a metalhydroxide, e.g., sodium or potassium hydroxide and the acylating agentadded. The reaction time can range from about 1 hour and about 4 hours,preferably about 2-2.5 hours. The temperature of the mixture ismaintained at a temperature generally ranging between about 5° C. andabout 70° C., preferably between about 10° C. and about 50° C. Theamount of alkali employed per equivalent of NH₂ groups in the aminoacids generally ranges between about 1.25 moles and about 3 moles, andis preferably between about 1.5 moles and about 2.25 moles perequivalent of NH₂. The pH of the reaction solution generally rangesbetween about pH 8 and about pH 13, and is preferably between about pH10 and about pH 12. The amount of amino modifying agent employed inrelation to the quantity of amino acids is based on the moles of totalfree NH₂ in the amino acids. In general, the amino modifying agent isemployed in an amount ranging between about 0.5 and about 2.5 moleequivalents, preferably between about 0.75 and about 1.25 equivalents,per molar equivalent of total NH₂ groups in the amino acids.

The modified amino acid formation reaction is quenched by adjusting thepH of the mixture with a suitable acid, e.g., concentrated hydrochloricacid, until the pH reaches between about 2 and about 3. The mixtureseparates on standing at room temperature to form a transparent upperlayer and a white or off-white precipitate. The upper layer isdiscarded, and modified amino acids are collected by filtration ordecantation. The crude modified amino acids are then mixed with water.Insoluble materials are removed by filtration and the filtrate is driedin vacuo. The yield of modified amino acids generally ranges betweenabout 30 and about 60%, and usually about 45%. The present inventionalso contemplates amino acids which have been modified by multipleacylation, e.g., diacylation or triacylation.

If amino acid esters or amides are the starting materials, they aredissolved in a suitable organic solvent such as dimethylformamide orpyridine, are reacted with the amino modifying agent at a temperatureranging between about 5° C. and about 70° C., preferably about 25° C.,for a period ranging between about 7 and about 24 hours. The amount ofamino modifying agents used relative to the amino acid esters are thesame as described above for amino acids.

Thereafter, the reaction solvent is removed under negative pressure andoptionally the ester or amide functionality can be removed byhydrolyzing the modified amino acid ester with a suitable alkalinesolution, e.g., 1 N sodium hydroxide, at a temperature ranging betweenabout 50° C. and about 80° C., preferably about 70° C., for a period oftime sufficient to hydrolyze off the ester group and form the modifiedamino acid having a free carboxyl group. The hydrolysis mixture is thencooled to room temperature and acidified, e.g., with an aqueous 25%hydrochloric acid solution, to a pH ranging between about 2 and about2.5. The modified amino acid precipitates out of solution and isrecovered by conventional means such as filtration or decantation.

The modified amino acids may be purified by acid precipitation,recrystallization or by fractionation on solid column supports.Fractionation may be performed on a suitable solid column supports suchas silica gel, alumina, using solvent mixtures such as aceticacid/butanol/water as the mobile phase; reverse phase column supportsusing trifluoroacetic acid/acetonitrile mixtures as the mobile phase;and ion exchange chromatography using water as the mobile phase. Themodified amino acids may also be purified by extraction with a loweralcohol such as methanol, butanol, or isopropanol to remove impuritiessuch as inorganic salts.

The modified amino acids generally are soluble in alkaline aqueoussolution (pH≧9.0); partially soluble in ethanol, n-butanol and 1:1 (v/v)toluene/ethanol solution and insoluble in neutral water. The alkalimetal salts, e.g., the sodium salt of the derivatized amino acids aregenerally soluble in water at about a pH of 6-8.

In poly amino acids or peptides, one or more of the amino acids may bemodified (acylated). Modified poly amino acids and peptides may includeone or more acylated amino acid(s). Although linear modified poly aminoacids and peptides will generally include only one acylated amino acid,other poly amino acid and peptide configurations can include more thanone acylated amino acid. Poly amino acids and peptides can bepolymerized with the acylated amino acid(s) or can be acylated afterpolymerization.

Special mention is made of the compound:

wherein A and B independently are Arg or Leu.

Sulfonated Amino Acid Complexing Perturbants

Sulfonated modified amino acids, poly amino acids, and peptides aremodified by sulfonating at least one free amine group with a sulfonatingagent which reacts with at least one of the free amine groups present.

Special mention is made of compounds of the formulaAr—Y—(R²⁴)_(n)—OH  LXIwherein Ar is a substituted or unsubstituted phenyl or naphthyl;Y is —SO₂—, R²⁴ has the formula

R²⁵ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl) phenyl, (C₁ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl,(C₁ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ toC₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀alkenyl);

R²⁵ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁to C₄ alkoxy, —OH, —SH and —CO₂R²⁷ or any combination thereof;

R²⁷ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R²⁵ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof; and

R²⁶ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Suitable, but non-limiting, examples of sulfonating agents useful inpreparing sulfonated amino acids include sulfonating agents having theformula R²⁸—SO₂—X wherein R²⁸ is an appropriate group for the modifiedamino acid being prepared such as, but not limited to, alkyl, alkenyl,cycloalkyl, or aromatics and X is a leaving group as described above.One example of a sulfonating agent is benzene sulfonyl chloride.

Modified poly amino acids and peptides may include one or moresulfonated amino acid(s). Although linear modified poly amino acids andpeptides used generally include only one sulfonated amino acid, otherpoly amino acid and peptide configurations can include more than onesulfonated amino acid. Poly amino acids and peptides can be polymerizedwith the sulfonated amino acid(s) or can be sulfonated afterpolymerization.

Modified Amino Acid Derivative Complexing Perturbants

Modified amino acid, polyamino acid, or peptide derivatives are aminoacids, poly amino acids, or peptides which have had at least oneacyl-terminus converted to an aldehyde or a ketone, and are acylated atat least one free amine group, with an acylating agent which reacts withat least one of the free amine groups present.

Amino acid, poly amino acid, or peptide derivatives can be readilyprepared by reduction of amino acid esters or peptide esters with anappropriate reducing agent. For example, amino acid, poly amino acid, orpeptide aldehydes can be prepared as described in an article by R. Chenet al., Biochemistry, 1979, 18, 921-926. Amino acid, poly amino acid, orpeptide ketones can be prepared by the procedure described in OrganicSyntheses. Col. Vol. IV, Wiley, (1963), page 5. Acylation is discussedabove.

For example, the derivatives may be prepared by reacting a single aminoacid, poly amino acid, or peptide derivative or mixtures of two or moreamino acid or peptide derivatives, with an acylating agent or an aminemodifying agent which reacts with free amino moieties present in thederivatives to form amides. The amino acid, poly amino acid, or peptidecan be modified and subsequently derivatized, derivatized andsubsequently modified, or simultaneously modified and derivatized.Protecting groups may be used to avoid unwanted side reactions as wouldbe known to those skilled in the art.

In modified poly amino acid or peptide derivative, one or more of theamino acid may be derivatized (an aldehyde or a ketone) and/or modified,(acylated) but there must be at least one derivative and at least onemodification.

Special mention is made of the modified amino acid derivativesN-cyclohexanoyl-Phe aldehyde, N-acetyl-Phe-aldehyde, N-acetyl-Tyrketone, N-acetyl-Lys ketone and N-acetyl-Leu ketone, and N-cyclohexanoylphenyl-alanine aldehyde.

Carboxylic Acid Complexing Perturbants

Various carboxylic acids and salts of these carboxylic acids may be usedas complexing perturbants. These carboxylic acids have the formula:R²⁹—CO₂H  LXII

wherein R²⁹ is C₁ to C₂₄ alkyl, C₂ to C₂₄ alkenyl; C₃ to C₁₀ cycloalkyl,C₃ to C₁₀ cycloalkenyl, phenyl, naphthyl, (C₁ to C₁₀ alkyl)phenyl, (C₂to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₂ to C₁₀alkenyl)naphthyl, phenyl(C₁ to C₁₀ alkyl), phenyl(C₂ to C₁₀ alkenyl),naphthyl(C₁ to C₁₀ alkyl) and naphthyl(C₂ to C₁₀ alkenyl);

R²⁹ being optionally substituted with C₁ to C₁₀ alkyl, C₂ to C₁₀alkenyl, C₁ to C₄ alkoxy, —OH, —SH, —CO₂R³⁰, C₃ to C₁₀ cycloalkyl, C₃ toC₁₀ cycloalkenyl, heterocyclic having 3-10 ring atoms wherein the heteroatom is one or more atoms of N, O, S or any combination thereof, aryl,(C₁ to C₁₀ alk)aryl, aryl(C₁ to C₁₀)alkyl, or any combination thereof;

R²⁹ being optionally interrupted by oxygen, nitrogen, sulfur, or anycombination thereof; and

R³⁰ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄ alkenyl.

The preferred carboxylic acids are cyclohexanecarboxylic acid,cyclopentanecarboxylic acid, cycloheptanecarboxylic acid, hexanoic acid,3-cyclohexanepropanoic acid, methylcyclohexanecarboxylic acid,1,2-cyclohexanedicarboxylic acid, 1,3-cyclohexanedicarboxylic acid,1,4-cyclohexanedicarboxylic acid, 1-adamantanecarboxylic acid,phenylpropanoic acid, adipic acid, cyclohexanepentanoic acid,cyclohexanebutanoic acid, pentylcyclohexanoic acid,2-cyclopentanehexanoic acid, cyclohexane pentanoic acid, hexanedioicacid, cyclohexanebutanoic acid, and (4-methylphenyl) cyclohexane aceticacid.

Other Examples of Complexing Perturbants

Although all complexing perturbants which can form the supramolecularcomplexes described herein are within the scope of the presentinvention, other examples of complexing perturbants include, but are notlimited to, 2-carboxymethyl-phenylalanine-leucine; 2-benzyl succinicacid, an actinonin, phenylsulfonyl aminophenyl-butyric acid,

Mimetics

Mimetics within the scope of the present invention are constructs whichare structural and/or functional equivalents of an original entity.Structural and chemically functional mimetics of the supramolecularcomplexes and the reversible transportable intermediate states of activeagents are not necessarily peptidic, as non-peptidic mimetics can beprepared which have the appropriate chemical and/or structuralproperties. However, preferred mimetics are peptides which have adifferent primary structure than the supramolecular complex or theintermediate state, but retain the same secondary and tertiary structureof the supramolecular complex or the intermediate state. Althoughmimetics may have less bioactivity than a native state or intermediatestate active agent or supra molecular comples, the mimetics may possessother important properties which may not be possessed by the nativestate such as, for example, further increased ability to be deliveredorally.

Methods of preparation of such mimetics are described, for example, inYamazaki et al., Chirality 3:268-276 (1991); Wiley et al.,Peptidomimetics Derived From Natural Products, Medicinal ResearchReviews, Vol. 13, No. 3, 327-384 (1993); Gurrath et al., Eur. J. Biochem210:991-921 (1992); Yamazaki et al, Int. J. Peptide Protein Res.37:364-381 (1991); Bach et al., Int. J. Peptide Protein Res. 38:314-323(1991); Clark et al., J. Med. Chem. 32:2026-2038 (1989); Portoghese, J.Med. Chem. 34:(6) 1715-1720 (1991); Zhou et al., J. Immunol. 149 (5)1763-1769 (Sep. 1, 1992); Holzman et al., J. Protein Chem. 10: (5)553-563 (1991); Masler et al., Arch. Insect Biochem, and Physiol.22:87-111 (1993); Saragovi et al., Biotechnology 10: (July 1992);Olmsteel et al., J. Med. Chem. 36:(1) 179-180 (1993); Malin et al.Peptides 14:47-51 (1993); and Kouns et al., Blood 80:(10) 2539-2537(1992); Tanaka et al., Biophys. Chem. 50 (1994) 47-61; DeGrado et al.,Science 243 (Feb. 3, 1989); Regan et al., Science 241: 976-978 (Aug. 19,1988); Matouschek et al, Nature 340: 122-126 (Jul. 13, 1989); Parker etal., Peptide Research 4: (6) 347-354 (1991); Parker et al., PeptideResearch 4:(6) 355-363 (1991); Federov et al., J. Mol. Biol. 225:927-931 (1992); Ptitsyn et al., Biopolymers 22: 15-25 (1983); Ptitsyn etal., Protein Engineering 2:(6) 443-447 (1989).

For example, protein structures are determined by the collective intra-and inter-molecular interactions of the constituent amino acids. Inalpha helices, the first and fourth amino acid in the helix interactnon-covalently with one another. This pattern repeats through the entirehelix except for the first four and last four amino acids. In addition,the side chains of amino acids can interact with one another. Forexample, the phenyl side chain of phenylaline would probably not besolvent exposed if that phenylalanine were found in a helix. If theinteractions of that phenylalanine contributed to helix stability thensubstituting an alanine for a phenylalanine would disrupt the helix andchange the conformation of a protein.

Therefore, a mimetic could be created by first determining which aminoacid side chains became solvent exposed and thus removed fromcontributing to stabilization of the native state such as by thetechnique of scanning mutagenesis. Mutants containing amino acidsubstitutions at those same sights could be created so that thesubstituted amino acids would render the protein conformation moreintermediate-like that native-like. Confirmation that the appropriatestructure had been synthesized could come from spectroscopy and otheranalytical methods.

Delivery Compositions

Delivery compositions which include the supramolecular complex describedabove are typically formulated by mixing the perturbant with the activeagent. The components can be prepared well prior to administration orcan be mixed just prior to administration.

The delivery compositions of the present invention may also include oneor more enzyme inhibitors. Such enzyme inhibitors include, but are notlimited to, compounds such as actinonin or epiactinonin and derivativesthereof. These compounds have the formulas below:

Derivatives of these compounds are disclosed in U.S. Pat. No. 5,206,384.Actinonin derivatives have the formula:

wherein R³¹ is sulfoxymethyl or carboxyl or a substituted carboxy groupselected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonylgroups; and R³² is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group.Other enzyme inhibitors include, but are not limited to, aprotinin(Trasylol) and Bowman-Birk inhibitor.

The delivery compositions of the present invention may be formulatedinto dosage units by the addition of one or more excipient(s),diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s),or dosing vehicle(s). Preferred dosage unit forms are oral dosage unitforms. Most preferred dosage unit forms include, but are not limited to,tablets, capsules, or liquids. The dosage unit forms can includebiologically, pharmacologically, or therapeutically effective amounts ofthe active agent or can include less than such an amount if multipledosage unit forms are to be used to administer a total dosage of theactive agent. Dosage unit forms are prepared by methods conventional inthe art.

The subject invention is useful for administering biologically activeagents to any animals such as birds; mammals, such as primates andparticularly humans; and insects. The system is particularlyadvantageous for delivering chemical or biologically active agents whichwould otherwise be destroyed or rendered less effective by conditionsencountered before the active agent in the native state reaches itstarget zone (i.e. the area to which the active agent to be delivered)and by conditions within the body of the animal to which they areadministered. Particularly, the present invention is useful in orallyadministering active agents, especially those which are not ordinarilyorally deliverable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the invention without limitation. Allparts and percentages are by weight unless otherwise indicated.

EXAMPLE 1 α-Interferon Native Gels

Native gradient gels (Pharmacia) were run with 647 μg/ml ofα-interferon, (Intron-A—Schering-Plough) and increasing amounts (10-500mg/mL) of perturbant (mixture of L-Valine, L-Leucine, L-phenylalanine,L-lysine and L-arginine modified with benzenesulfonylchloride)(valine—7.4%, leucine—16.5%, phenylalanine—40.3%, lysine—16.2% andarginine—19.6%). 4 μl of material were loaded onto the gel using a 6/4comb for loading.

Results are illustrated in FIG. 1.

-   Lane 1=High molecular weight marker (Bio-Rad)−1:20 dilution    w/dH₂O−(5 μl−>100 μl).-   Lane 2=α-interferon A (647 μg/mL) control 5 μl+5 μl Bromophenol Blue    (BPB)−(1.29 μg loaded).-   Lane 3=α-interferon+perturbant (10 mg/mL)−50 μl α-interferon+50 μl    BPB=100 μl (1.29 μg loaded).-   Lane 4=α-interferon+perturbant (50 mg/mL) 50 μl α-interferon+50 μl    BPB=100 μl (1.29 μg loaded).-   Lane 5=α-interferon+perturbant (100 mg/mL ) 50 μl α-interferon+50 μl    BPB=100 μl (1.29 μg loaded).-   Lane 6=α-interferon+perturbant (500 mg/mL) 5 μl α-interferon+5 μl    BPB=10 μl (1.29 μg loaded).

EXAMPLE 1A α-Interferon Native Gradient Gel

The method of Example 1 was followed substituting the thermalcondensation product of glutamic acid, aspartic acid, tyrosine, andphenyl-alanine (Glu-Asp-Tye-Phe) that was fractionated through a 3000molecular weight cut-off filter for the perturbant.

Results are illustrated in FIG. 2.

Samples

-   Lane 1=High Molecular Weight marker (Bio-Rad).-   Lane 2=α-interferon (647 μg/mL)−5 μl+5 μl BPB control.-   Lane 3=α-interferon+perturbant (10 mg/mL)−50 μl+50 μl BPB=100 μl.-   Lane 4=α-interferon+perturbant−50μl+50 μl BPB=100 μl.-   Lane 5=α-interferon+perturbant (100 mg/mL)−50 μl Intron A+50 μl    BPB=100 μl.-   Lane 6=α-interferon+perturbant (500 mg/mL)−5 μl Intron A+50 μl    BPB=100 μl.

Examples 1 and 1 A illustrate that α-interferon alone (lane 2 in FIGS. 1and 2) banded at the appropriate molecular weight (approximately 19,000Daltons). As the amount of perturbant added is increased in eachsubsequent lane relative to a fixed concentration of α-interferon, theα-interferon migrates to a lower, rather than a higher, molecularweight. The change seen with the perturbant of Example 1 is morepronounced than that seen with the perturbant of Example 1A. Thisindicates that the α-interferon structure is changing due to the twodifferent perturbants, because if structure were not changing, therewould be a shift towards higher molecular weight as perturbant complexeswith the active agent.

EXAMPLE 2 Oral Administration of α-interferon and Perturbant to Rats

Male Sprague-Dawley rats (average weight approximately 250 mg) werefasted overnight on wire racks with no bedding. Prior to dosing, animalswere anesthetized with a combination ketamine/thorazine subcutaneous.Dosing solutions of the composition prepared according to Example 1 at500 μg/kg were administered via oral gavage through a 10-12 cm rubbercatheter attached to a 1 cc syringe containing the dosing solution.Blood samples were drawn by tail vein bleeding at the designated timepoints. Serum was prepared and frozen at −70° C. until ready for assay.Serum samples were assayed by ELISA (Biosource International, Camarillo,Calif., Cytoscreen Immunoassay Kit™, Catalog # ASY-05 for human IFN-α).

Results are illustrated in FIG. 3.

EXAMPLE 2A Oral Administration of α-Interferon and Perturbant to Rats

The method of Example 2 was followed substituting a dosing solution ofthe composition prepared according to Example 1A at 78 μg/kg. Resultsare illustrated in FIG. 3.

COMPARATIVE EXAMPLE 2* Oral Administration of α-Interferon to Rats

α-interferon at 100 μg/kg without perturbant was administered accordingto the procedure of Example 2.

Results are illustrated in FIG. 3.

EXAMPLE 3 Oral Administration of Salmon Calcitonin and Perturbant toRats

The perturbant of Example 1 was reconstituted with distilled water andadjusted to a pH of 7.2-8.0 with HCl or NaOH. Salmon calcitonin (sCt)was dissolved in a citric acid stock solution (0.085 N and then combinedwith the perturbant solution to obtain the final dosing solution. Finalconcentrations of perturbant and SCt were 400 mg/mL and 5 μg/mLrespectively.

Results are illustrated in Table 1 below.

24 hour fasted male Sprague Dawley rats weighing 100-150 g wereanesthetized with ketamine. Rats were administered the dosing solutionin a vehicle by oral gavage at 800 mg/kg of perturbant and 10 μg/kg ofsCt. The dosing solution was administered using a 10 cm rubber catheter.One hour post-dosing, the rats were administered 1.5 mg/kg thorazine and44 mg/kg ketamine by intramuscular injection. At 1, 2, 3, and 4 hourspost-dosing, blood samples were drawn from the rat tail artery for serumcalcium concentration determination using the Sigma Diagnostic Kit(Catalog # 587-A, Sigma Chemical Co, St. Louis, Mo.).

Results are illustrated in FIG. 4.

EXAMPLE 3A Oral Administration of Salmon Calcitonin and Perturbant toRats

The method of Example 3 was followed substituting L-tyrosine modified bycyclohexanoyl chloride as the perturbant.

Results are illustrated in FIG. 4.

COMPARATIVE EXAMPLE 3*

Salmon calcitonin (10 μg/kg) without perturbant was administered to ratsaccording to the procedure of Example 3.

Results are illustrated in FIG. 4.

EXAMPLE 4 Isothermal Titration Calometry

A dosing composition of the perturbant of Example 1 at 2.4 mM and sCt at0.3 mM was prepared, and isothermal titration calorimetry was performedat pH 6.5 and 4.5. The buffer at pH 6.5 was 30 mM Hepes-30 mM NaCl, andthe buffer at pH 4.5, was 30 mM sodium acetate-30 mM NaCl.

All experiments were performed at 30° C. using 8.0 mM perturbant in thedropping syringe and 1.0 mM calcitonin in the calorimeter cell. In allexperiments, 15×10 μl increments of perturbant were added in 10 secondduration additions with 2 minutes equilibration between additions.

Results were validated in experiments where perturbant (8 mM) was placedin the dropping syringe, and equivalent increments were added to pH 4.5buffer (no sCt) and where perturbant was placed in the dropping syringeand 10 μl increments were added to pH 6.5 buffer (no sCt). Titrationcurves were not obtained in these experiments, and the results showedthat heat of mixing and/or dilution of perturbant is negligible.Therefore, the experimental isotherms were not corrected by backgroundsubtraction.

Results are illustrated in Table 1 below.

EXAMPLE 4A

The method of Example 4 was followed substituting the perturbant ofExample 1A. Results were validated in experiments where perturbant wasplaced in the dropping syringe, and equivalent increments were added topH 4.5 buffer (no sCt).

Results are illustrated in Table 1 below.

TABLE 1 Binding Parameters of Perturbants as Determined by ITC¹ K_(D) ΔHΔS (M) (cal/mol) (cal/mol ° K.) N pH 6.6 Example 4 4.59 × 10⁻⁸ +240+34.4 0.6 Example 4A 6.99 × 10⁻⁹ +277 +38.3 11.6 pH 4.5 Example 4Precipitates Example 4A 1.29 × 10⁻⁴ +553 +19.8 +0.8 ¹Calorimetryexperiments were performed essentially as detailed by You, J. L.,Scarsdale, J. N., and Harris, R. B., J. Prot. Chem. 10: 301-311, 1991;You, Junling, Page, Jimmy D., Scarsdale, J. Neel, Colman, Robert W., andHarris, R. B., Peptides 14: 867-876, 1993; Tyler-Cross, R., Sobel, M.,Soler, D. F., and Harris, R. B., Arch. Biochem. Biophys. 306: 528-533,1993; Tyler-Cross, R., Sobel, M., Marques, D., and Harris, R. B.,Protein Science 3: 620-627, 1994.

EXAMPLE 5 GuHCl Denaturation Of α-Interferon

A stock solution of 9.1 mg/mL of α-interferon (Schering Plough Corp.) in20 mM sodium phosphate buffer at pH 7.2 was prepared. Samples wereprepared by diluting the α-interferon with the sodium phosphate bufferand 10 M guanidine hydrochloride (GuHCl) (Sigma Chemical Co.—St. Louis,Mo.) stock solution to 200 ug/mL concentration of α-interferon atvarious concentrations of GuHCl. Diluted samples were allowed to come toequilibrium by incubation for approximately 30 minutes at roomtemperature prior to measurement.

Fluorescence measurements were made at 25° C. using a Hitachi F-4500.Protein tryptophan fluorescence was observed at an excitation wavelengthof 298 nm and an emission wavelength of 343 nm. ANS(1-anilinonapthalene-8-sulfonate) fluorescence was observed at anexcitation wavelength of 355 nm and an emission wavelength of 530 nm.For all fluorescence measurements, a 5 nm spectral bandpass was chosenfor both excitation and emission.

Results are illustrated in FIG. 5.

EXAMPLE 6 Concentration Effect of GuHCl on α-Interferon Configuration

GuHCl 5M stock solution was prepared using 20 mM sodium phosphate, pH7.2 buffer. After dilution, the pH of the stock was checked and adjustedby concentrated HCl. To determine the concentration of final solutionthe refractive index referenced in Methods in Enzymology, Vol. 6, page43 by Yasuhiko Nozaki was used.

α-interferon stock (9.1 mg/mL) was mixed with sufficient amounts ofGuHCl to yield the concentrations of Table 1A below:

TABLE 1A α-lnterferon/GuHCl Solutions GuHCl (M) α-IFN (mg/mL) 0.5 0.601.0 0.53 1.5 0.60 2.0 0.50 3.0 0.60 4.0 0.50

Differential scanning calorimetry (DSC) was run, and results areillustrated in FIG. 6.

EXAMPLE 7 pH Titration of Intron A as Measured by Intrinsic TryptophanFluorescence

A stock solution of 9.1 mg/mL α-interferon in 20 mM sodium phosphatebuffer at pH 7.2 (Schering Plough Corp.) was prepared. Samples wereprepared by diluting the α-interferon to a concentration of 200 ug/mLinto solution buffered at various pH values using the following buffers:Glycine at pH 2 and 12, sodium phosphate at pH 3, 4, 5, 7, and boricacid at pH 8. These buffers were prepared as described in the PracticalHandbook of Biochemistry and Molecular Biology, Edited by Gerald D.Fasman, 1990. Diluted samples were allowed to come to equilibrium byincubation for approximately 30 minutes at room temperature prior tomeasurement.

Fluorescence was measured according to the procedure of Example 5.Results are illustrated in FIG. 7.

EXAMPLE 8 ph Titration of Insulin Measured by ANS Fluorescence

A stock solution was prepared by dissolving 2 mg of insulin in 1 mL ofdeionized water. 1-anilinonaphthalene-8-sulphonate (ANS) stock solutionwas prepared by dissolving 10 mg in 10 mL of deionized water. Sampleswere prepared by diluting the insulin to a concentration of 200 ug/mLinto solution buffered at various pH values using the following buffers:Glycine at pH 2 and 12, sodium phosphate at pH 3, 4, 5, 7, and boricacid at pH 8. These buffers were prepared as described in the PracticalHandbook of Biochemistry and Molecular Biology, Edited by Gerald D.Fasman, 1990. The final ANS concentration was 90 ug/mL. Diluted sampleswere allowed to come to equilibrium by incubation for approx. 30 minutesat room temperature prior to measurement.

Fluorescence was measured according to the procedure of Example 5.Results are illustrated in FIG. 8.

EXAMPLE 9 Reversibility of Circular Dichroism Spectra of α-interferon atpH 2 and 7.2

Circular dichroism spectra of α-interferon were generated at pH 7.2. ThepH of the solution was then readjusted to pH 2, and the sample wasrescanned. The sample solution was then readjusted to 7.2 and rescanned.

Concentration of α-interferon was 9.2 μM or 0.17848 mg/mL, ([IFN]stock=9.1 mg/mL). Buffers used were 20 mM NaPhosphate at pH 7.2; and 20mM Glycine at pH 2.0.

Reversal of the pH to 7.2 resulted in complete restoration of the nativestructure, demonstrating the reversibility of the intermediate state. Itis believed that the free energy difference between the native state andthe intermediate state is small.

Results are illustrated in FIGS. 9A and 9B.

EXAMPLE 10 Circular Dichroism Spectra of α-Interferon at 7.2—pHDependence

The extent of ordered secondary structure of α-interferon at differentpH's was determined by circular dichroism (CD) measurements in the farUV range. The large dilution factor of interferon stock (^(˜)50 times)resulted in the sample being at the proper pH. Concentration ofα-interferon was 9.2 μM or 0.17848 mg/mL, ([IFN] stock=9.1 mg/mL).Buffers used were 20 mM sodium phosphate at pH 6.0 and 7.2; 20 mM NaAcat pH 3.0, 4.0, 4.5, 5.0 and 5.5; and 20 mM Glycine at pH 2.0

The secondary structure content was estimated with several fittingprograms, each of which decomposes the CD curve into four majorstructural components: α-helix, β-sheet, turns, and random coil. Two ofthose programs were provided with the CD instrument as an analysisfacility. The first program uses seven reference proteins: Myoglobin,Lysozyme, Papain, Cytochrome C, Hemoglobin, Ribonuclease A andChymotrypsin. The second uses Yang.REF reference file.

A third program, CCAFAST, uses the Convex Constraint Algorithm and isdescribed in “Analysis of Circular Dichroism Spectrum of Proteins Usingthe Convex Constraint Algorithm: A Practical Guide”. (A. Perczel, K.Park and G. D. Fasman (1992) Anal. Biochem. 203: 83-93).

Deconvolution of the far UV scans over a range of pH volumes (2.0-7.2)indicates significant compaction of the secondary structure at pH 3.5.The near UV scan indicates a disruption of tertiary structure packing,and the far UV scan indicates that there is still significant secondarystructure at this pH.

Results are illustrated in FIG. 10.

EXAMPLE 11 DSC of Insulin and Increasing Concentrations of GuHCl

DSC was performed with 6 mg/mL insulin (0.83 mM assuming a molecularweight of 6,000) in 50 mM phosphate buffer, pH 7.5. Each subsequentthermogram was corrected by background subtraction of a 0.6Mguanidine-phosphate buffer solution.

Insulin was freshly prepared as a concentrated stock solution in 50 mMphosphate buffer, pH 7.5, and an appropriate aliquot was diluted inbuffer, filtered though a 2 micron PTFE filter, and degassed for atleast 20 minutes. The reference cell contained degassed buffer.

Scanning calorimetry was performed using 5 mg 0.83 mM porcine insulin(MW 6,000) per mL in 50 mM phosphate buffer, pH 7.5. All thermogramswere performed on a Microcal MC-2 scanning calorimeter equipped with theDA2 data acquisition system operated in the upscale mode at 1° C./min(up to 90° C.), and data points were collected at 20 second intervals.All scans were initiated at least 20 degrees below the observedtransitions for the active agent. All thermograms were corrected forbaseline subtraction and normalized for the concentration ofmacromolecule. According to the methods of the Johns HopkinsBiocalorimetry Center, See, for example, Ramsayetal. Biochemistry (1990)29:8677-8693; Schon et al. Biochemistry (1989) 28:5019-5024 (1990) 29:781-788. The DSC data analysis software is based on the statisticalmechanical deconvolution of a thermally induced macromolecular meltingprofile.

The effect of GuHCl on structure was assessed in DSC experiments whereindividual solutions were prepared in phosphate buffer, pH 7.5,containing denaturant diluted from a 5M stock solution to concentrationsranging for 0.5-2M.

Results are illustrated in Table 2 below.

TABLE 2 DSC of Insulin and Increasing Concentrations of GuanidineHydrochloride$\frac{{Tm}\quad\left( {{Cp},\max} \right)}{\left( {{^\circ}\quad{C.}} \right)}$Insulin 0.0 M GuHCl 78.3 Insulin + 0.5 M GuHCl 79.3 Insulin + 1.0 MGuHCl 77.5 Insulin + 2.0 M GuHCl 69.7 Insulin + 3.0 M GuHCl notransition observed

EXAMPLE 12 Effect of Ionic Strength on the DSC Spectrum of Insulin

A sample containing 6 mg/mL insulin (0.83 mM in 50 mM phosphate buffer,pH 7.5, containing 0.25, 0.5, or 1.0M NaCl) was used. Thermograms wereperformed according to the procedure in Example 11 and were corrected bysubtraction of a 0.5M NaCl-phosphate buffer blank as described above.

The effect of increasing ionic strength on structure was assessed in DSCexperiments where individual solutions were prepared so as to containNaCl at concentrations ranging from 0.25-3M.

Results are illustrated in Table 3 below.

TABLE 3 Effect of Ionic Strength on the DSC Spectrum of Insulin$\frac{{Tm}\quad\left( {{Cp},\max} \right)}{\left( {{^\circ}\quad{C.}} \right)}$Insulin 0.0 M NaCl 78.3 Insulin + 0.25 M NaCl 80.7 Insulin + 0.5 M NaCl80.7 Insulin + 1.0 M NaCl 80.7

EXAMPLE 12A Effect of Ionic Strength on the DSC Spectrum of rhGh

The method of Example 11 was followed substituting 5 mg/mL recombinanthuman growth hormone (rhGh) (225 μM based on M, 22, 128 of HGH) in 50 mMphosphate buffer, pH 7.5 containing either 0.5 or 1.0M NaCl, for theinsulin. The thermograms were corrected by subtraction of a 0.5MNaCl-phosphate buffer blank.

Results are illustrated in Table 4 below.

TABLE 4 Effect of Ionic Strength on the DSC Spectrum of rhGh$\frac{{Tm}\quad\left( {{Cp},\max} \right)}{\left( {{^\circ}\quad{C.}} \right)}$$\frac{\Delta H}{\left( {{kcal}/{mol}} \right)}$ rhGh 0.0 M NaCl 75.2191.0 rhGh + 0.5 M NaCl 75.8 89.7 rhGh + 10.0 M NaCl 76.5 50.5

EXAMPLE 13 Effect of pH on the DSC Spectrum of rhGH

5 mg/mL rhGh were dissolved in buffer (0.17 mM in 50 mM phosphatebuffer, assuming a molecular weight of 20,000). The pH of the solutionwas adjusted to the desired value, and all curves were corrected bybaseline subtraction.

The effect of pH on structure was assessed by DSC according to theprocedure of Example 11 where individual solutions were prepared inphosphate buffer ranging in pH value from 2.0 to 6.0.

Results are illustrated in Table 5 below.

TABLE 5 Effect of pH on the DSC Spectrum of rhGh$\frac{{Tm}\quad\left( {{Cp},\max} \right)}{\left( {{^\circ}\quad{C.}} \right)}$$\frac{\Delta H{^\circ}}{\left( {{kcal}/{mol}} \right)}$ pH 2.0 notransition observed no transition observed pH 3.0 no transition observedno transition observed pH 3.5 no transition observed no transitionobserved pH 4.0 ≈73.0 broad transition pH 5.0 75.0 161 pH 6.0 75.2 191pH 7.5 (10 mg/mL) a) 73 (a) + (b) = 632 b) 75

EXAMPLE 14 Effect of GuHCl on the DSC Spectrum of rhGh

An initial scan of rhGh was performed at 10 mg/mL in the absence ofGuHCl (0.33 mM assuming 20,000 molecular weight). Subsequently, theconcentration of rhGh was lowered to 5 mg/mL (0.17 mM) in 50 mMphosphate buffer, pH 7.5 containing varying concentrations of GuHCl.Each subsequent thermogram was corrected by background subtraction of a0.5M guanidine-phosphate buffer solution. The thermograms were correctedby subtraction of a 0.5M NaCl-phosphate buffer blank. Scans wereperformed according to the procedure of Example 11.

Results are illustrated in Table 6 below.

TABLE 6 Effect of Guanidine Hydrochloride on DSC Spectrum of rhGh$\frac{\begin{matrix}{{DOMAIN}\quad A} \\{{Tm}\quad\left( {{Cp},\max} \right.}\end{matrix}}{\left( {{^\circ}\quad{C.}} \right)}\quad$$\frac{\begin{matrix}{{DOMAIN}\quad B} \\{{Tm}\quad\left( {{Cp},\max} \right)}\end{matrix}}{\left( {{^\circ}\quad{C.}} \right)}\quad$$\frac{\Delta H}{\left( {{kcal}/{mol}} \right)}$ rhGh 72.6 74.3 632rhGh + 0.5 M GuHCl 71.5 not defined, 48 but present rhGh + 1.0 M GuHCl70.9 absent 109 rhGh + 1.5 M GuHCl 69.7 absent 12 rhGh + 2.0 M GuHCl70.0 absent 58 rhGh + 2.5 M GuHCl 70.7 absent 99

EXAMPLE 15 pH Dependence of α-Interferon Conformation

α-interferon stock (9.1 mg/mL) was diluted with buffer to aconcentration of 0.6 mg/mL. The sample was dialyzed overnight in buffer(volume ratio of α-interferon to buffer was 1:4000). Since there was noextinction coefficient provided, concentration of the sample used wasdetermined by comparison of absorption spectra of the sample before andafter dialysis. For each particular pH, the absorbance of thenondialyzed α-interferon of known concentration was measured at 280 nm.Then after dialysis, absorbance was read again to account for theprotein loss, dilution, etc. Buffer conditions and α-interferonconcentrations were:

-   pH 3.0: Buffer—20 mM NaAc. [IFN]=0.50 mg/mL;-   pH 4.1: Buffer—20 mM NaAc. [IFN]=0.53 mg/mL;-   pH 5.0: Buffer—20 mM NaAc. [IFN]=0.37 mg/mL;-   pH 6.0: Buffer—20 mM Na Phosphate. [IFN]=0.37 mg/mL;-   pH 7.2: Buffer—20 mM Na Phosphate. [IFN]=0.48 mg/mL.-   DSC scans were performed according to the procedure of Example 11.    Although clear, transparent solutions of α-interferon were obtained    for every pH at room temperature, there were noticeable signs of    precipitation at pH 5.0 and 6.0 after the temperature scans.    -   Results are illustrated in Table 7 below.

TABLE 7 α-Interferon - pH dependence DSC pH Tm° C. ΔH cal/mol 7.2 66.84732717 6.0 65.34  45580 5.0 67.32  69782 4.1 65.64  60470 3.0 — —

EXAMPLE 16 Concentration Effect of GuHCl on α-Interferon Conformation

GuHCL/α-interferon samples were prepared according to the method ofExample 6. DSC scans were performed according to the procedure ofExample 11.

Results are illustrated in Table 8 below.

TABLE 8 α-Interferon in GuHCl DSC [GuHCl] M Tm° C. ΔH cal/mol 0.0 67.1272562 0.5 64.43 50827 1.0 63.04 41705 1.5 60.11 29520 2.0 56.32 249803.0 45.90 20577 4.0 — —

Examples 5-16 illustrate that ionic strength, guanidine hydrochlorideconcentration, and pH result in changes in the Tm of active agents,indicating a change in conformation. This was confirmed by fluorescencespectroscopy. The reversible intermediate conformational states can beused as templates to prepare mimetics.

EXAMPLE 17 Preparation of α-Interferon Intermediate State Mimetics

An intermediate conformational state of α-interferon is determined. Apeptide mimetic having the secondary and tertiary structure of theintermediate state is prepared.

EXAMPLE 18 Preparation of Insulin Intermediate State Mimetics

The method of Example 17 is followed substituting an insulin for theα-interferon.

EXAMPLE 19 Preparation of rhGh Intermediate State Mimetics

The method of Example 17 is followed substituting recombinant humangrowth hormone for the α-interferon.

EXAMPLE 20 In vivo Administration of α-Interferon Mimetics

Rats are dosed according to the procedure of Example 2 with the mimeticprepared according to the procedure of Example 17.

EXAMPLE 21 In vivo Administration of Insulin Mimetics

The procedure of Example 20 was followed, substituting the mimeticprepared according to the procedure of Example 18.

EXAMPLE 22 In vivo Administration of rhGH Mimetics

The procedure of example 19 was followed, substituting the mimeticprepared according to the procedure of Example 19.

EXAMPLE 24 Titration of α-Interferon as Measured by Intrinsic TryptophanFluorescence

A stock solution of 9.1 mg/mL α-interferon in 20 mM sodium phosphatebuffer at pH 7.2 was prepared. A stock solution of perturbant wasprepared by dissolving 800 mg of perturbant (L-arginine acylated withcyclohexanoyl chloride) in 2 mL of 20 mM Sodium Phosphate buffer (pH 7).

Samples were prepared by diluting the α-interferon with the sodiumphosphate buffer and perturbant stock solution at various perturbantconcentrations. Diluted samples were allowed to come to equilibrium byincubation for approximately 30 minutes at room temperature prior tomeasurement.

Fluorescence from the endogenous tryptophan resident of α-interferonwere measured according to the procedure of Example 5. The perturbantdid not contain a fluoophore.

Results are illustrated in FIG. 11.

EXAMPLE 25 In vivo Administration of Perturbant and α Interferon to Rats

Rats were dosed according to the method of Example 2 with dosingsolutions containing the perturbant of Example 24 (800 mg/kg) mixed withα-interferon (1 mg/kg). Serum samples were collected and assayed byELISA according to the procedure of Example 2.

Results are illustrated in FIG. 12.

COMPARATIVE EXAMPLE 25*

Rats were dosed according to the method of Example 25 with α-interferon(1 mg/kg). Serum samples were collected and assayed according to theprocedures of Example 25.

Results are illustrated in FIG. 12.

EXAMPLE 26 Differential Scanning Colorimetry of α-Interferon andPerturbant

Perturbant binding DSC was conducted using 20 mM NaPhosphate buffer atpH 7.2. Dry perturbant was weighed out to make perturbant stocksolutions. α-interferon stock was diluted in the buffer. α-interferonsolution was not dialyzed prior to experiments for the purpose of havingthe same active concentration for the whole set.

DSC thermograms were generated with α-interferon at a concentration of0.64 mg/ml and a perturbant (phenylsulfonyl-para-aminobenzoic acidpurified to >98% (as determined by reverse phase chromatography prior togeneration of the spectra)) at perturbant concentrations of 5, 10, 25and 100 mg/ml. DSC was conducted on a DASM-4 differential scanningcalorimeter interfaced to an IBM PC for automatic collection of thedata. The scanning rate was 60° C./h.

Results are illustrated in Table 9 below and FIG. 13.

COMPARATIVE EXAMPLE 26* Different Scanning Calorimetry of α-Interferon

The method of Example 26 was followed substituting α-interferon withoutperturbant. Results are illustrated in Table 9 below and FIG. 13.

TABLE 9 α-lnterferon + Perturbant DSC Perturbant-mg/ml Tm° C. ΔH cal/mol0 67.12 72562 5 64.37 60151 10 62.3 53161 25 58.15 35393 100 46.185439.3

DSC scans where the added concentration of perturbant ranged from 0-100mg/mL show induced conformational changes in the α-interferon that occurin a concentration dependent manner. At 100 mg/mL of the perturbant, thethermogram indicated that the α-interferon Cp vs. Tm curve was a flatline. The flat Cp vs. Tm curve obtained at 100 mg/mL of perturbantindicates that hydrophobic residues within the αinterferon moleculebecame solvent exposed. It is clear that the perturbant was able tochange the structure of α-interferon in a concentration. dependentmanner.

EXAMPLE 27 Dialysis Experiments—Reversibility of Complexing with thePerturbant

An α-interferon stock solution at a concentration of 9.1 mg/mL wasdiluted with buffer to an α-interferon concentration of 0.6 mg/mL. DSCwas performed according to the procedure of Example 26.

Results are illustrated in FIG. 14A.

α-interferon (0.6 mg/ml) and the perturbant of Example 26 (100 mg/ml)were mixed with no apparent changes in the Cp of the solution. Thissolution was then dialyzed overnight into phosphate buffer, and thethermogram was rerun. Results are illustrated in FIG. 14B.

The dialyzed sample had essentially the same Tm and the same area underthe Cp vs. Tm curve as it did prior to addition of the perturbant. Thisindicated that not only was the perturbant able to induce conformationalchanges in the protein but that this process was reversible. Dilutionwas enough of a driving force to effect disengagement of the perturbantfrom the active agent.

EXAMPLE 28 Perturbant and α-Interferon DSC

The method of Example 6 was followed, substituting the perturbant ofExample 26 for the GuHCl.

Results are illustrated in FIG. 15.

The DSC experiments on the equilibrium denaturation of α-interferonindicate the existence of intermediate conformations of the molecule. ΔHvs. Tm plots indicate the energetics of intermediate conformationsoccupied by α-interferon at each set of experimental conditions.

EXAMPLE 29 In Vivo Administration of Perturbant and α Interferon to Rats

Rats were dosed according to the method of Example 2 with a dosingsolution of the perturbant of Example 4 (800 mg/kg) and α-interferon (1mg/kg). Serum samples were collected and assayed by ELISA according tothe procedure of Example 2.

Results are illustrated in FIG. 16.

COMPARATIVE EXAMPLE 29* In Vivo Administration of α Interferon to Rats

Rats were dosed according to the method of Example 29 with α-interferon(1 mg/kg) without perturbant. Serum samples were collected and assayedaccording to the procedure of Example 29.

Results are illustrated in FIG. 16.

FIG. 16 illustrates that when active agent mixed with perturbant wasorally gavaged into animals, significant serum titers of α-interferonwere detectable in the systemic circulation, and the α-interferon wasfully active. Confirming data that the delivered α-interferon was fullyactive included the fact that the serum was assayed by a commercialELISA kit which utilizes a monoclonal antibody able to recognize anepitope specific to the native conformation of Intron and that the serumwas further assayed using the cytopathic effect assay which determinedtiters of Intron that correlated with the titers measured by ELISA (datanot shown). Therefore, the conformational changes which occurred as aresult of with the perturbant, were reversible changes.

EXAMPLE 30 Perturbant Concentration Dependent Change in α-Interferon

The method of Example 26 was followed substituting cyclohexanoylphenylglycine for the perturbant.

Results are illustrated in Table 10 below and in FIG. 17.

TABLE 10 α-Interferon + Perturbant DSC Perturbant mg/ml Tm° C. ΔHcal/mol 0 67.12 72562 5 63.00 42299 10 59.49 43058 25 52.79 27237 100 —0

Cyclohexanoyl phenylglycine induced conformational changes inα-interferon that were concentration dependent.

EXAMPLE 31 In Vivo Administration of Perturbant and α-Interferon to Rats

Rats were dosed according to the method of Example 2 with dosingsolutions containing the perturbant of Example 30 (800 mg/kg) andα-interferon 1 (mg/kg). Serum samples were collected and assayed byELISA according to the procedure of Example 2.

Results are illustrated in FIG. 18.

COMPARATIVE EXAMPLE 31* In vivo Administration of Perturbant andα-Interferon to Rats

Rats were dosed according to the method of Example 2 with α-interferon(1 mg/kg) without perturbant. Serum samples were collected and assayedby ELISA according to the procedure of Example 2.

Results are illustrated in FIG. 18.

EXAMPLE 32 Perturbant and α-Interferon DSC

The method of Example 6 was followed substituting the perturbant ofExample 30 for the GuHCl.

Results are illustrated in FIG. 19.

The ΔH v. Tm plot indicates the existence of an equilibrium intermediateconformation of α-interferon that is stable at below 5 and 25 mg/ml ofadded cyclohexanoyl phenylglycine perturbant.

EXAMPLE 33 Isothermal Titration Calorimetry of α-Interferon withPerturbant

Isothermal titration calorimetry of perturbant complexing withα-interferon was performed at 25° C. at two different pH's. The buffersused were 20 mM NaPhosphate for pH 7.2 and 20 mM NaAc for pH 3.0.α-interferon solution was dialyzed before the experiment to reach theappropriate pH. Dry perturbants were weighed and diluted in dialysate.

ITC was conducted on a MicroCal OMEGA titration calorimeter (MicroCalInc.—Northampton, Mass.). Data points were collected every 2 seconds,without subsequent filtering. α-interferon solution placed in 1.3625 mLcell was titrated using a 250 μL syringe filled with concentratedperturbant solution. A certain amount of titrant was injected every 3-5minutes for up to 55 injections.

A reference experiment to correct for the heat of mixing of twosolutions, was performed identically except that the reaction cell wasfilled with buffer without active agent.

Analysis of the data was performed using the software developed at theJohns Hopkins University Biocalorimetry Center.

The titration at pH 7.2 included 53 injections of 2 μL of the perturbantof Example 30 (50 mg/mL=191.6 mM (MW 261)) and α-interferon (1.3mg/mL=0.067 mM (MW 19400)).

Results are illustrated in FIG. 20.

Curve fitting indicated multiple independent sites:

-   n (1)=121.0354 where n=# of completed perturbant molecules-   ΔH (1)=58.5932 cal/Mole perturbant-   log 10 Ka (1)=2.524834 where Ka=association constant-   x-axis units are concentration of carrier in mM.-   y-axis units represent heat/injection expressed in calories.

At pH 3, complexing resulted in a negative enthalpy.

COMPARATIVE EXAMPLE 33* Isothermal Titration Calorimetry of Perturbant

The method of Example 33 was followed, substituting 53 injections of 2μl for the perturbant (50 mg/mL=191.6 mM) [IFN]=0 mg of Example 30without active agent.

Example 33 and Comparative Example 33 illustrate that α-interferon has apositive enthalpy and a binding constant (K_(d)≈10⁻³M).

EXAMPLE 34 Isothermal Titration Calorimetry of α-Interferon andPerturbant Complexing

The method of Example 33 was followed substituting the perturbant ofExample 26 for the perturbant of Example 30.

The titration at pH 7.2 included two runs of 55 injections each of 5 μLof perturbant (50 mg/mL=181 mM (FW 277)) and α-interferon (2.31mg/mL=0.119 mM, (MW 19400)).

Results are illustrated in FIG. 21.

Curve fitting indicated multiple independent sites:

-   n (1)=55.11848 where n=# of complex perturbant molecules-   ΔH (1)=−114.587 cal/Mole perturbant-   log 10 Ka (1)=2.819748 where Ka=association constant-   x-axis units are concentration of carrier in mM.-   y-axis units represent heat/injection expressed in calories.

Complexing of perturbant to α-interferon at pH 3.0 resulted inprecipitation of the complex out of the solution. Due to the heat effectproduced by this process, it was impossible to measure the complexingparameters.

COMPARATIVE EXAMPLE 34* Isothermal Titration Calorimetry of Perturbant

The method of Example 34 was followed, substituting 55 injections of 5μl of the perturbant of Example 26(50 mg/mL=181 mM) in 20 mM sodiumphosphate pH 7.2 without active agent.

The perturbant of Example 26 complexed with α-interferon resulted in anegative enthalpy and a comparable binding constant to that of theperturbant of Example 30 and α-interferon.

Examples 33 and 34 indicate that the stronger the perturbant complexeswith the active agent and the more thermodynamically stable theintermediate state of the active agent, the greater the bioavailabilityof the active agent.

Therefore, by plotting the ΔH v. Tm curve for an active agent and aperturbant, those perturbants that induce little or no enthalpic changeover the broadest range of Tm would be preferred perturbants. It isbelieved that perturbants that stabilize the intermediate state to agreater extent will result in more efficient delivery of the activeagent.

EXAMPLE 35 Comparison of the Effects of Three Perturbants on ΔH vs. TmPlots with α-Interferon

DSC experiments were carried out according to the procedure of Example26, with 0.5 mg/ml α-interferon mixed with (1) benzoyl para-aminophenylbutyric acid, (2) the perturbant of Example 30, or (3) theperturbant of Example 26.

Benzoyl para-amino phenylbutyric acid was poorly soluble under thebuffer conditions. Maximum concentration at which the solution was stilltransparent at room temperature was ^(˜)8 mg/mL. Therefore, theconcentrations of the perturbant used were 2, 4, and 6 mg/mL. Resultsare illustrate in FIGS. 22 and 23. The dashed line in FIG. 22 representsthe linear least squares, and the regression equation is at the top ofFIG. 22.Y=−1.424 e ⁵+3148.8xR=0.9912

FIGS. 22 and 23 illustrate that conformational changes in α-interferonare more readily produced by benzoyl para-amino phenylbutyric acid thanby the perturbants of Examples 30 and 26, and that such changes are morereadily produced by the perturbant of Example 30 than by the perturbantof Example 26.

EXAMPLE 36 Isothermal Titration Calorimetry of α-Interferon andComplexing

ITC was performed according to the method of Example 33 with 40injections of 5 μL of the perturbant benzoyl para-amino phenylbutyricacid (7.5 mg/mL=24.59 mM, (FW 305)) and α-interferon (2.5 mg/mL=0.129mM, (MW 19400)).

Results are illustrated in FIG. 24.

Curve fitting indicated multiple independent sites:

-   n (1)=23.69578 where n=# of complexed perturbant molecules-   ΔH (1)=791.5726 cal/Mole perturbant-   log 10 Ka (1)=3.343261 where Ka=association constant-   x-axis represents concentration of carrier in mM.-   y-axis represents heat/injection expressed in calories.

COMPARATIVE EXAMPLE 36*

ITC was performed according to the method of Example 36 with 40injections of 5 μl of the perturbant benzoyl para-amino phenylbutyricacid (7.5 mg/mL=24.59 mM) in 20 mM NaPhosphate pH 7.2 buffer, withoutactive agent.

The apparent dissociation constant for the perturbant of Example 35 isgreater than that for the perturbant of Example 30 (10⁻⁴M) at pH 7.

Therefore, benzoyl para-amino phenylbutyric acid complexes more stronglyto α-interferon and induces the native state-reversible intermediateconformational state at lower concentrations of perturbant.

EXAMPLES 37-39 Comparative In Vivo Pharmacokinetics of VariousPerturbants and α-Interferon

Rats were dosed according to the procedure of Example 2 with dosingsolutions containing the perturbant of Example 26 (800 mg/kg) (1), theperturbant of Example 30 (800 mg/kg) (2), or the perturbant of benzoylpara-amino phenylbutyric acid (300 mg/kg) (3) and α-interferon at 1mg/kg. Serum samples were collected and assayed by ELISA according tothe procedure of Example 2.

Results are illustrated in FIG. 25.

COMPARATIVE EXAMPLE 37* In Vivo Pharmacokinetics of α-Interferon

Rats were dosed according to the method of Example 37 with α-interferonwithout perturbant. Serum samples were collected and assayed by ELISAaccording to the procedure of Example 2.

Results are illustrated in FIG. 25.

Examples 35-39 illustrate that in vivo potency was correctly predictedby in vitro modeling.

EXAMPLES 40-42 Comparative In Vivo Pharmacokinetics of VariousPerturbants with rhGh in Hypophysectomized Rats

Rats were dosed according to the procedure of Example 2, with dosingsolutions containing the perturbants salicyloyl chloride modifiedL-phenylalanine (1.2 g/kg) (40), phenylsulfonyl para-amino benzoic acid(1.2 g/kg) (41), or cyclohexanoyl chloride modified L-tyrosine (1.2g/kg) (42) mixed with rhGh (1 mg/kg).

Rats were hypophysectomized according to the procedure of Loughna, P. T.et al, Biochem. Biophys. Res. Comm., Jan. 14, 1994, 198(1), 97-102.Serum samples were assayed by ELISA (Medix Biotech, Inc., Foster City,Calif., HGH Enzyme Immunoassay Kit).

Results are illustrated in FIG. 26.

EXAMPLES 43-45 Isothermal Titration Calorimetry of rhGH at pH 7.5 and4.0 with Different Perturbants

The ability of rhGh to complex with various perturbants was assessed byITC using a Microcal Omega titrator, usually equilibrated at 30° C. Thesample cell of the calorimeter was filled with degassed rhGH (usually at0.25 mM) prepared in 50 mM phosphate buffer, pH 7.5 or 4.0. Theperturbant (cyclohexanoyl chloride modified L-tyrosin (a), salicyloylmodified L-phenylalanine (b), or phenylsulfonyl-para-amino benzoic acid(c)) was then placed in the dropping syringe at 1 mM (for pH 7.5) and2.5 mM, (for pH 4.0). Twenty to twenty-five 10 μl injections were madeinto rapidly mixing (400 rpm) solution with 2 minute intervals betweeninjections.

Initial concentration of perturbant placed in the calorimeter samplecell assumed a formula weight of 200 for each perturbant. The pH of eachsolution was checked after dissolution, but no adjustments of the pHwere required. All experiments were performed at 30° C. Initialconcentration of rhGh placed in the dropping syringe assumed a molecularweight of 20,000 for rhGh. The pH of each solution was checked afterdissolution, but no adjustment of the pH was required.

The heats of reaction were determined by integration of the observedpeaks. To correct for heat of mixing and dilution, a control experimentwas also performed under identical conditions where aliquots of the testperturbant or rhGh were added to buffer solution only. The sum total ofthe heat evolved was plotted against the total perturbant concentrationto produce the isotherm from which the association constant (K_(A), M),enthalpy change (ΔH, kcal/mol), entropy change (ΔS (eu), and N, and thestoichiometry of perturbant molecules complexed per equivalent ofcomplexed supramolecular complex, were determined by curve-fitting thebinding isotherm against the binding equation described for perturbantcomplexing in a supramolecular complex possessing one set of independentperturbant complexing sites. The data were deconvoluted using thenonlinear least squares algorithm supplied in the software of themanufacturer.

Results are illustrated in Table 11 below.

TABLE 11 Isothermal Titration Calorimetry of rhGH at pH 7.5 and 4.0 withDifferent Perturbants rhGh K_(D) ΔH ΔS Perturbant (mM) (M) (kcal/mol)(eu) N pH 7.5 A at 0.25 mM 1.0 9.88 × 10⁻⁵ +1.4 +23.5 7.0 B at 0.25 mM1.0 1.11 × 10⁻⁶ +2.1 +35.0 0.7 C at 0.25 mM 1.0 1.11 × 10⁻⁹ +0.8 +44.010.0 pH 4.0 A at 0.25 mM 1.0 7.81 × 10⁻⁵ −1.5 − 5.6 2.3 B at 0.25 mM 1.01.61 × 10⁻⁹ −35.6 −90.0 155.9 C at 0.25 mM 1.0 2.67 × 10⁻⁸ −1.2 −30.0122.0 A = cyclohexanoyl chloride modified L-tyrosine B = salicyloylmodified L-phenylalanine C = phenylsulfonyl-para-aminobenzoic acid

The positive ΔS values at pH 7.5 indicate that complexing at this pHresults in structural change.

EXAMPLES 46 AND 47 Pancreatin Inhibition Assay with α-Interferon andPerturbants

The assay for pancreatin activity was prepared as follows: 0.1 mL of astock solution of α-interferon (9.1 mg/mL, 20 mM NaH₂PO₄, pH 7.2)(Schering-Plough Corp.) was added to 2.5 mL of eitherphenylsulfonyl-para-aminobenzoic acid perturbant (46) or cyclohexanoylphenylglycine perturbant (47) (200 mg/mL) in 5 mM KH₂PO₄, pH 7.0.Incubation was carried out at 37° C. for 30 and 60 minutes following theaddition of 0.1 mL of USP pancreatin (20 mg/mL) (Sigma Chemical Co.) 0.1mL aliquots were withdrawn at those times points. Enzyme reactions werestopped by the addition of protease inhibitors (Aprotinin andBowman-Birk Inhibitor (BBI), each at 2 mg/mL) and were diluted five-foldto quantitate α-interferon left intact. A reverse phase HPLC methodusing a Butyl C-4 cartridge (3.0×0.46 cm, Rainin) and employing gradientelution between 0.1% TFA/water and 90% ACN in 0.1% TFA coupled with UVdetection at 220 nm was used for separating and quantitatingα-interferon. The α-interferon at 0 minutes was quantitated from analiquot prior to the addition of pancreatin and was taken to be 100%.

Results are illustrated in FIG. 27.

Examples 46 and 47 illustrate that both supramolecular complexesresisted enzymatic degradation. However, in additional testing nocorrelation was shown between the enzyme inhibitors potency and theability to deliver drug.

EXAMPLE 48 DSC of Heparin at pH 5.0

DSC thermograms of heparin at pH 5.0 were conducted according to themethod of Example 11 using pH, GuHCl, and ionic strength as perturbants.

Thermograms were corrected by subtraction of a heparin 0.05MNaCl—phosphate buffer blank, but an individual blank was not used foreach NaCl concentration.

Results are illustrated in Tables 12-14 below and in FIG. 28.

TABLE 12 Effects of pH on the DSC Spectrum of 20 μg/ml Heparin in 50 mMPhosphate Buffer Tm (Cp, max)$\frac{\Delta H}{\left( {{kcal}/{mol}} \right)}$$\frac{{\Delta H}_{vH}}{\left( {{kcal}/{mol}} \right)}$ pH 6.0 62.5232.1 13.8 pH 6.5 (a) 62.7 213.9 (b) 71.8 751.9 56.8 pH 7.0 (a) 47.1187.1 (b) 72.9 136.4 27.6 pH 7.5 66.2 499.4 83.8 (a) = a domain (b) = bdomain

TABLE 13 Effects of 10 M Guanidine Hydrochloride in 50 mM PhosphateBuffer on the DSC Spectrum of Heparin Tm (Cp, max)$\frac{\Delta H}{\left( {{kcal}/{mol}} \right)}$$\frac{{\Delta H}_{vH}}{\left( {{kcal}/{mol}} \right)}$ heparin 67.2499.4 83.8 heparin + 0.5 M GuHCl 50.5 287.3 170.9 heparin + 1.0 M GuHCl60.5 415.0 97.1 heparin + 1.5 M GuHCl — 1716.5 24.3 heparin + 2.0 MGuHCl — 2533.7 19.2

TABLE 14 Effect of Ionic Strength on the DSC Spectrum of 20 μg/ml ofHeparin in 50 mM Phosphate Buffer pH 7.0 Tm (Cp, max)$\frac{\Delta H}{\left( {{kcal}/{mol}} \right)}$$\frac{{\Delta H}_{vH}}{\left( {{kcal}/{mol}} \right)}$ 0.0 M NaCl 47.1187.1 72.9 136.4 0.25 M NaCl 46.1 0.112 not present 0.50 M NaCl 41.60.094 not present 0.75 M NaCl 27.5 0.00 not present 1.0 M NaCl notransition observed

These data indicate that non-proteinaceous active agents are able tochange conformation in response to a perturbant.

EXAMPLE 49 Column Chromatography of Heparin and Perturbants

The following materials were used:

Column:

-   10 mm×30 cm, low pressure, glass column from Pharmacia w/adjustable    bed volume. The bed volume used was 22 cm at a pressure of 0.8 Mpa.    Packing:-   Heparin covalently bonded to Sepharose CL-6B with no linker    molecule.-   Sepharose fractionation range: 10,000-4,000,000.-   The density of heparin was 2 mg/cc as per Pharmacia Q.C. Department.    Conditions:-   The mobil phase was 67 mM phosphate buffer, pH 7.4.-   The flow rate was 1.5 mL/min isocratic.-   The run time was 45 minutes.-   Sample detection was done with a Perkin Elmer refractive index    detector.

Column integrity was confirmed by injecting protamine and observing aretention time greater than 1 hour. Void volume was determined byinjecting water and measuring time of elution.

Each of the perturbants of Table 15 below (5 mg) was independentlydissolved in 1 mL of mobil phase and injected (100 ul) into the column.Time of elution was measured. K′ value was determined by using thefollowing equation (as per USP):K′=(Ret. time Carrier/Ret. time Water)−1

The results were compared between each perturbant as well as theirrespective in vivo performance in FIG. 29. K′ (the degree ofretardation) values in the figure have been corrected by subtraction ofthe K′ value determined from the sepharose column from the K′ valuedetermined from the heparin-sepharose column.

TABLE 15 PERTURBANTS cyclohexylidenebutyric acid (2)-Na salt #1cylcohexanebutyroyl (2-) aminobutyric acid (4) #2phenylacetyl-para-aminobutyric acid #3ortho-methylcyclohexanoyl-aminobutyric acid (4) #4phenylacetyl-aminohexanoic acid (6-) #5cinnamoyl-para-aminophenylbutyric acid #6 cyclohexanebutyroyl(2-)-para-aminophenylbutyric acid #7hydrocinnamoyl-para-aminophenylbutyric acid #8 cyclohexanebutyroyl(2-)-leu-leu #9 cyclohexanebutyroyl (2-)-gly #10

EXAMPLE 50 Oral Administration of Heparin to Rats

Rats were dosed with the dosing solutions of Table 16 below according tothe procedure of Example 2. Blood was collected, and activated partialthromboplastin time (APTT) was performed as described in Henry, J. B.,Clinical Diagnosis and Management by Laboratory Methods, W. B. Saunders,1979.

Results are illustrated in FIGS. 29 and 30.

FIG. 29 illustrates that as predicted in the model, the greater thebinding to heparin the greater the elevation of APTT. The data suggeststhat at K′ values below 0.2, activity is likely to be poor. At K′values >0.2, activity will be significant.

The data indicate a correlation between the retardation by the heparinsepharose column relative to just a sepharose column and the increasedin vivo activity as measured by elevation of APTT. Notably protamine,which binds most strongly to heparin, has no oral bioavailability(K′=3.68). This indicates that balancing binding strength andconformational changes with the ability to dissociate will optimize thefull complement of biological activity of the drug.

TABLE 16 HEPARIN/PERTURBANTS DOSING SCHEDULES Solution 1 =cinnamoyl-para-aminophenylbutyric acid pH 7.5, N = 5 Solution 2 =cinnamoyl-para-aminophenylbutyric acid (300 mg/kg) + Heparin (100 mg/kg)in propylene glycol/water (1:1, pH 7.4) Solution 3 − Heparin (100 mg/kg,pH 7.4, N = 5) Solution 4 = hydrocinnamoyl-para-aminophenylbutyric acid(300 mg/kg) + Heparin (100 mg/kg) in propylene glycol/water (1:1, pH7.4)

EXAMPLE 51 Comparison of the Effects of Six Perturbants On ΔH vs. TmPlots with DPPC

DSC experiments were carried out according to the procedure of Example26, with 1.0 mg/ml dipalmitoylphosphatidylcholine (DPPC) mixed withperturbants XI, L, LII, LIII, and LIV. The concentrations of theperturbant were varied from 0 to 20 mg/ml.

Results are illustrated in FIG. 31.

EXAMPLE 52 Differential Scanning Colorimetry of DPPC and PerturbantCompound L

Perturbant binding DSC was conducted using 20 mM NaPhosphate buffer atpH 7.2. Dry perturbant L was weighed out to make perturbant stocksolutions. DPPC stock solution was prepared in the buffer.

DSC thermograms were generated with DPPC at a concentration of 1.0mg/ml. The perturbant concentrations used were 0, 5, 10, and 20 mg/ml.The DSC was performed as described in Example 26.

Results are illustrated in FIG. 32.

EXAMPLE 53 Differential Scanning Colorimetry of DPPC Perturbant CompoundL and rhGH

Perturbant binding DSC was conducted using 20 mM NaPhosphate buffer atpH 7.2. Dry perturbant L was weighed out to make perturbant stocksolutions. DPPC stock solution was prepared in the buffer. rhGH solutionwas prepared as described in Example 12A.

DSC thermograms were generated with DPPC at a concentration of 1.0mg/ml. Samples having DPPC alone; DPPC with 10 mg/ml of perturbant; DPPCwith 0.3 mg/ml of rhGH; and DPPC, 10 mg/ml of perturbant and 0.3 mg/mlof rhGH were prepared and analyzed. The DSC was performed as describedin Example 26.

Results are illustrated in FIG. 33.

EXAMPLE 54 Differential Scanning Colorimetry of DPPC Perturbant CompoundLII and rhGH

Perturbant binding DSC was conducted using 20 mM NaPhosphate buffer atpH 7.2. Dry perturbant LII was weighed out to make perturbant stocksolutions. DPPC stock solution was prepared in the buffer. rhGH solutionwas prepared as described in Example 12A.

DSC thermograms were generated with DPPC at a concentration of 1.0mg/ml. Samples having DPPC alone; DPPC with 5 mg/ml of perturbant; DPPCwith 0.3 mg/ml of rhGH; and DPPC, 5 mg/ml of perturbant and 0.3 mg/ml ofrhGH were prepared and analyzed. The DSC was performed as described inExample 26.

Results are illustrated in FIG. 34.

EXAMPLE 55 Differential Scanning Colorimetry of DPPC Perturbant CompoundXI and rhGH

Perturbant binding DSC was conducted using 20 mM NaPhosphate buffer atpH 7.2. Dry perturbant XI was weighed out to make perturbant stocksolutions. DPPC stock solution was prepared in the buffer. rhGH solutionwas prepared as described in Example 12A.

DSC thermograms were generated with DPPC at a concentration of 1.0mg/ml. Samples having DPPC alone; DPPC with 2 mg/ml of perturbant; DPPCwith 0.3 mg/ml of rhGH; and DPPC, 2 mg/ml of perturbant and 0.3 mg/ml ofrhGH were prepared and analyzed. The DSC was performed as described inExample 26.

Results are illustrated in FIG. 35.

EXAMPLE 55 Dynamic Light Scattering of Compound L

Solutions of compound L were prepared in a 10 mM phosphate buffer at apH of 7.0. The concentrations tested were 10, 15, and 20 mg/ml. Thesolutions were analyzed using standard microscopic light scatteringtechniques.

Results are illustrated in FIG. 36.

All patents, applications, test methods, and publications mentionedherein are hereby incorporated by reference.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed disclosure. Forexample, oral drug delivery entails crossing from the lumen of thegastrointestinal tract to the blood. This occurs as a result of crossingseveral cellular lipid bilayers that separate these anatomicalcompartments. The complexation of the perturbant with the active agentand the change in conformation of the active agent creates asupramolecular complex having physicochemical properties, such as, forexample, solubility and conformation in space, which are different thanthose of either the perturbant or the active agent alone. This suggeststhat one can take advantage of this property to cross other membranessuch as the blood-brain barrier, and ophthalmic, vaginal, rectal, andthe like membranes. All such modifications are within the full extendedscope of the appended claims

1. A method for preparing a composition, said method comprising mixing:(A) at least one biologically-active agent; and (B) a compound havingthe formula:Ar—Y—(R¹⁴)_(n)—OH wherein: Ar is a substituted or unsubstituted phenylor naphthyl,

R¹⁵ has the formula

R¹⁵ is C₁ to C₂₄ alkyl, C₂ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl) phenyl, (C₂ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl,(C₂ to C₁₀ to alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₂ toC₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl), and naphthyl (C₂ to C₁₀alkenyl); R¹⁵ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄alkenyl, C₁ to C₄ alkoxy, —OH, —SH, —CO₂R¹⁷, cycloalkyl, cycloalkenyl,heterocyclic alkyl, alkaryl, heteroaryl, heteroalkaryl, or anycombination thereof; R¹⁷ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄alkenyl; R¹⁵ is optionally interrupted by oxygen, nitrogen, sulfur orany combination thereof; and R¹⁶ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄alkenyl; and n is from 1 to
 5. 2. An oral delivery compositioncomprising: (a) a biologically active agent in an intermediateconformational state non-covalently complexed with (b) a complexingperturbant having the formula:Ar—Y—(R¹⁴)_(n)—OH wherein: Ar is a substituted or unsubstituted phenylor naphthyl;

R¹⁴ has the formula

R¹⁵ is C₁ to C₂₄ alkyl, C₂ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl) phenyl, (C₂ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl,(C₂ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₂ toC₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl), and naphthyl (C₂ to C₁₀alkenyl); R¹⁵ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄alkenyl, C₁ to C₄ alkoxy, —OH, —SH, —CO₂R¹⁷, cycloalkyl, cycloalkenyl,heterocyclic alkyl, alkaryl, heteroaryl, heteroalkaryl, or anycombination thereof; R¹⁷ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄alkenyl; R¹⁵ is optionally interrupted by oxygen, nitrogen, sulfur orany combination thereof; and R¹⁶ is hydrogen, C₁ to C₄ alkyl or C₂ to C₄alkenyl; and n is from 1 to
 5. 3. The composition according to claim 2,wherein Ar is substituted or unsubstituted phenyl, Y is

—C—, R¹⁵ is C₁ to C₂₄ alkyl, (C₁ to C₁₀ alkyl) phenyl; or phenyl (C₁ toC₁₀ alkyl) and n is equal to
 1. 4. The composition according to claim 2,wherein said biologically-active agent is a pharmacological agent or atherapeutic agent.
 5. The composition according to claim 2, wherein saidbiologically-active agent is selected from the group consisting of apeptide, a polysaccharide, a mucopolysaccharide, a carbohydrate, alipid, a pesticide or any combination thereof.
 6. The compositionaccording to claim 2, wherein said biologically-active agent is apeptide.
 7. The composition according to claim 6, wherein saidbiologically-active agent is a hormone.
 8. The composition according toclaim 2, wherein said biologically-active agent is a mucopolysaccharide.9. The composition according to claim 2, wherein saidbiologically-active agent is a lipid.
 10. The composition according toclaim 2, wherein said biologically-active agent is selected from thegroup consisting of human growth hormone, bovine growth hormone, growthhormone-releasing hormone, an interferon, interleukin-I, interleukin-II,insulin, heparin, low molecular weight heparin, calcitonin,erythropoetin, atrial naturetic factor, an antigen, a monoclonalantibody, somatostatin, adrenocorticotropin, gonadotropin releasinghormone, oxytocin, vasopressin, cromolyn sodium, vancomycin,desferrioxamine, an anti-fungal, an anti-microbial agent or anycombination thereof.
 11. The composition according to claim 10, whereinsaid biologically-active agent is selected from the group consisting ofan interferon, insulin, human growth hormone, heparin, low molecularweight heparin, calcitonin, cromolyn sodium, anti-fungal, and ananti-microbial agent.
 12. The composition according to claim 10, whereinsaid biologically-active agent is an interferon.
 13. The compositionaccording to claim 10, wherein said biologically-active agent isinsulin.
 14. The composition according to claim 10, wherein saidbiologically-active agent is human growth hormone.
 15. The compositionaccording to claim 10, wherein said biologically-active agent isheparin.
 16. The composition according to claim 15, wherein saidbiologically-active agent is low molecular weight heparin.
 17. Thecomposition according to claim 10, wherein said biologically-activeagent is calcitonin.
 18. The composition according to claim 10, whereinsaid biologically-active agent is cromolyn sodium.
 19. The compositionaccording to claim 10, wherein said biologically-active agent is ananti-microbial.
 20. A dosage unit form comprising (A) a compositionaccording to claim 2, and (B) (a) an excipient, (b) a diluent, (c) adisintegrant, (d) a lubricant, (e) a plasticizer, (f) a colorant, (g) adosing vehicle, or (h) any combination thereof.
 21. A dosage unit formaccording to claim 20, comprising a tablet, a capsule, or a liquid. 22.A method for administering a biologically-active agent to an animal inneed of said agent said method comprising administering orally to saidanimal the composition as defined in claim 2.